Substituted tetracycline compounds

ABSTRACT

The present invention pertains, at least in part, to novel substituted tetracycline compounds. These tetracycline compounds can be used to treat numerous tetracycline compound-responsive states, such as bacterial infections and neoplasms.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/876,313, filed on Dec. 21, 2006 and U.S. Provisional PatentApplication No. 60/943,003, filed Jun. 8, 2007. The contents of theforegoing applications are hereby incorporated in their entirety.

BACKGROUND OF THE INVENTION

The development of the tetracycline antibiotics was the direct result ofa systematic screening of soil specimens collected from many parts ofthe world for evidence of microorganisms capable of producingbactericidal and/or bacteriostatic compositions. The first of thesenovel compounds was introduced in 1948 under the name chlortetracycline.Two years later, oxytetracycline became available. The elucidation ofthe chemical structure of these compounds confirmed their similarity andfurnished the analytical basis for the production of a third member ofthis group in 1952, tetracycline. A new family of tetracyclinecompounds, without the ring-attached methyl group present in earliertetracyclines, was prepared in 1957 and became publicly available in1967; and minocycline was in use by 1972.

Recently, research efforts have focused on developing new tetracyclineantibiotic compositions effective under varying therapeutic conditionsand routes of administration. New tetracycline analogues have also beeninvestigated which may prove to be equal to or more effective than theoriginally introduced tetracycline compounds. Examples include U.S. Pat.Nos. 2,980,584; 2,990,331; 3,062,717; 3,165,531; 3,454,697; 3,557,280;3,674,859; 3,957,980; 4,018,889; 4,024,272; and 4,126,680. These patentsare representative of the range of pharmaceutically active tetracyclineand tetracycline analogue compositions.

Historically, soon after their initial development and introduction, thetetracyclines were found to be highly effective pharmacologicallyagainst rickettsiae; a number of gram-positive and gram-negativebacteria; and the agents responsible for lymphogranuloma venereum,inclusion conjunctivitis, and psittacosis. Hence, tetracyclines becameknown as “broad spectrum” antibiotics. With the subsequent establishmentof their in vitro antimicrobial activity, effectiveness in experimentalinfections, and pharmacological properties, the tetracyclines as a classrapidly became widely used for therapeutic purposes. However, thiswidespread use of tetracyclines for both major and minor illnesses anddiseases led directly to the emergence of resistance to theseantibiotics even among highly susceptible bacterial species bothcommensal and pathogenic (e.g., pneumococci and Salmonella). The rise oftetracycline-resistant organisms has resulted in a general decline inuse of tetracyclines as antibiotics of choice.

SUMMARY OF THE INVENTION

In one embodiment, the invention pertains, at least in part, tosubstituted tetracycline compounds of Formula I:

wherein

X is CHC(R¹³Y′Y), CR^(6′)R⁶, C═CR^(6′)R⁶, S, NR⁶, or O;

E is NR^(7d)R^(7e), OR^(7f) or (CH₂)₀₋₁C(═W′)WR^(7g);

W is O, S, NR^(7h) or CR^(7i)R^(7j);

W′ is O, S or NR^(7k);

R², R^(2′), R^(4′), R^(4a) and R^(4b) are each independently hydrogen,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety;

R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁴ is NR^(4a)R^(4b), alkyl, alkenyl, alkynyl, hydroxyl, halogen, orhydrogen;

R⁵ and R^(5′) are each hydroxyl, hydrogen, thiol, alkanoyl, aroyl,alkaroyl, aryl, heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkylcarbonyloxy, or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(7g), R^(7h), R^(7i),R^(7j) and R^(7k) are each independently hydrogen, allyl, alkyl,alkenyl, alkynyl, hydroxyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, amino, alkylamino, aminoalkyl, acyl, aryl, arylalkyl,alkylcarbonyloxy, alkylcarbonyloxylalkyl or arylcarbonyloxy, or R^(7c)and R^(7d) or R^(7e) and R^(7f) are linked to form a ring;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R⁹ is hydrogen, nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, aminoalkyl, amido, arylalkenyl, arylalkynyl, thionitroso, or(CH₂)₀₋₃(NR^(9c))₀₋₁—C(═Z′)ZR^(9a);

Z is CR_(9d)R^(9e), S, NR^(9b) or O;

Z′ is O, S, or NR^(9f);

R^(9a), R^(9b), R^(9c), R^(9d), R^(9e) and R^(9f) are each independentlyhydrogen, acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,heteroaromatic or a prodrug moiety;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, amido, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts thereof.

In another embodiment, the invention pertains, at least in part, tosubstituted tetracycline compounds of Formula II:

wherein

X is CHC(R¹³Y′Y), CR^(6′)R⁶, C═CR^(6′)R⁶, S, NR⁶, or O;

J is NR^(7m)R^(7n), OR^(7o) or heteroaryl;

R², R^(2′), R⁴, R^(4a) and R^(4b) are each independently hydrogen,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety;

R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a prodrug moiety;

R⁴ is NR^(4a)R^(4b), alkyl, alkenyl, alkynyl, hydroxyl, halogen, orhydrogen;

R⁵ and R^(5′) are each independently hydroxyl, hydrogen, thiol,alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, alkyl carbonyloxy, or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R^(7l), R^(7m), R^(7n) and R^(7o) are each independently hydrogen,alkyl, alkenyl, alkynyl, hydroxyl, alkoxy, amino, alkylamino,aminoalkyl, acyl, alkylthio, alkylsulfinyl, alkylsulfonyl, aryl,arylalkyl, alkylcarbonyloxy, or arylcarbonyloxy;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R⁹ is hydrogen, nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, aminoalkyl, amido,arylalkenyl, arylalkynyl, thionitroso or—(CH₂)₀₋₃(NR^(9c))₀₋₁C(═Z′)ZR^(9a);

Z is CR^(9d)R^(9e), S, NR^(9b) or O;

Z′ is O, S, or NR^(9f);

R^(9a), R^(9b), R^(9c), R^(9d), R^(9e) and R^(9f) are each independentlyhydrogen, acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,heteroaromatic or a prodrug moiety;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, amido, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts thereof.

In yet another embodiment, the present invention pertains, at least inpart, to substituted tetracycline compounds of Formula III:

wherein

R^(7p) is acyl, alkylamino, or heteroaryl;

R^(10a) is hydrogen, aryl, carboxylate or alkoxycarbonyl;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; andpharmaceutically acceptable salts thereof.

In another embodiment, the present invention pertains, at least in part,to substituted tetracycline compounds of Formula IIIa:

wherein

R^(4p) is —N(CH₃)₂ or hydrogen;

R^(7p′) is hydrogen, amino, acyl, heteroaryl, aminoalkyl;

R^(10a′) is hydrogen, heteroaryl, alkoxycarbonyl, carboxylate, cyano,alkyl or alkoxy; and pharmaceutically acceptable salts thereof.

The present invention also pertains, at least in part, to substitutedtetracycline compounds of Formula IV;

wherein

L is O, NH or SH;

K is N or CR^(7p′);

M is N or CR^(7p″):

R^(7p′) is hydrogen;

R^(7p″) is hydrogen, aminoalkyl or alkoxycarbonylaminoalkyl, andpharmaceutically acceptable salts thereof.

In yet another embodiment, the present invention pertains, at least inpart, to substituted tetracycline compounds of Formula IVa:

wherein

L* is O, NH or S;

*M is N, CH or CR^(7ps″);

R^(7ps″) is aminoalkyl; and pharmaceutically acceptable salts thereof.

In another embodiment, the present invention pertains, at least in part,to substituted tetracycline compounds of Formula V:

wherein

R⁴ is hydrogen;

R^(4′) is hydrogen or alkylamino;

R^(9f) is CR^(9g)NR^(9h) or CR^(9i)R^(9j)NR^(9k)R^(9l);

R^(9g), R^(9h), R^(9i), R^(9j), R^(9k) and R^(9l) are each independentlyhydrogen, alkyl, hydroxyl, amino, urea or alkoxy, or R^(9k) and R^(9l)are joined to form a ring; and pharmaceutically acceptable saltsthereof.

In another embodiment, the present invention pertains, at least in part,to substituted tetracycline compounds of Formula Va:

wherein

Q is —CH₂ or —C═CH₂;

R⁴ is hydrogen;

R^(4′) is hydrogen or alkylamino;

R^(5a′) is hydrogen or hydroxyl;

R^(7qa) is —N(CH₃)₂ or hydrogen;

R^(9q) is CR^(9g′) NR^(9h′) or CR^(9i′)R^(9j′)NR^(9k′)R^(9l′);

R^(9g′), R^(9h′), R^(9i′), R^(9j′) NR^(9k′) and R^(9l′) independentlyhydrogen, alkyl, hydroxyl, amino, urea or alkoxy, or R^(9k′) and R^(9l′)are joined to form a ring; and pharmaceutically acceptable saltsthereof.

In one embodiment, the present invention pertains, at least in part, tosubstituted tetracycline compounds of Formula VI:

wherein

X is CHC(R¹³Y′Y), CR^(6′)R⁶, C═CR^(6′)R⁶, S, NR⁶, or O;

p is a single bond or a double bond;

Q is CR^(7s) when p is a double bond or Q is CR^(7s′) R^(7s″) when p isa single bond;

R², R^(2′), R^(4′), R^(4a) and R^(4b) are each independently hydrogen,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, aryl alkyl, aryl, heterocyclic,heteroaromatic or a prodrug moiety;

R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁴ is NR^(4a)R^(4b), alkyl, alkenyl, alkynyl, hydroxyl, halogen, orhydrogen; R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl,aryl, heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷, R^(7s′) and R^(7s″) are each hydrogen, alkyl, alkenyl, alkynyl,hydroxyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, amino,aminoalkyl, alkylamino, aryl, acyl, arylalkyl, alkyl carbonyloxy, orarylcarbonyloxy;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R⁹ is hydrogen, nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, aminoalkyl, amido,arylalkenyl, arylalkynyl, thionitroso or—(CH₂)₀₋₃(NR^(9c))₀₋₁C(═Z′)ZR^(9a);

Z is CR^(9d)R^(9e), S, NR^(9b) or O;

T is O, S, or NR^(W):

R^(9a), R^(9b), R^(9c), R^(9d), R^(9e) and R^(9f) are each independentlyhydrogen, acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,heteroaromatic or a prodrug moiety;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, amido, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts thereof.

In a further embodiment, the present invention pertains, at least inpart, to substituted tetracycline compounds of Formula VII:

wherein

X is CR^(6′)R⁶;

R⁵ is hydroxyl or hydrogen;

R^(5′) is hydrogen;

R^(6′) hydrogen or alkyl;

R⁶ is hydrogen;

R^(7r) is hydrogen or alkylamino;

R^(9m) is heteroaryl, aminocarbonyl, hydroxyaminocarbonyl,alkoxyaminocarbonyl or —CR^(9m) NR^(9m′);

R^(9m′) and R^(9m″) are each hydrogen, hydroxy, alkyl, alkenyl, alkynyl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl; and pharmaceutically acceptable salts thereof.

In yet another embodiment, the present invention pertains, at least inpart, to substituted tetracycline compounds of Formula VIIa:

wherein

X is CR^(6*′)R^(6*);

R^(5*) is hydrogen;

R^(5*′) is hydrogen hydroxyl;

R^(6*′) is hydrogen or alkyl;

R^(6*) is hydrogen;

R^(7r*) is hydrogen, alkyl, heteroaryl, acyl or alkylamino;

R^(9m*) is aminoalkyl, heterocyclic, aryl, —CONR^(9ma)R^(9mb);—COR^(9m*′), —COOR^(9m*′), alkyl, cycloalkyl or hydrogen;

R^(9m′*) is aminoalkyl, aryl or alkyl;

R^(9m*″) is alkyl, alkoxyalkyl or hydroxyalkyl;

R^(9ma) and R^(9mb) are each hydrogen, hydroxyl, alkyl, hydroxyalkyl,aryl or alkoxy or are linked to form a ring; and pharmaceuticallyacceptable salts thereof.

The present invention also pertains, at least in part, to substitutedtetracycline compounds of Formula VIII:

wherein:

T is N or CR^(7td);

R⁴ is alkylamino or hydrogen;

R^(4′) is hydrogen;

R^(7te) is hydrogen.

R^(7ta), R^(7tb), R^(7tc) and R^(7td) are each independently hydrogen,halogen, hydroxyalkyl, hydroxyalkylaminocarbonyl, alkylaminoalkyloxy,aminocarbonyl, alkylaminoalkylaminocarbonyl, aminoalkylaminocarbonyl,methylpipcrazinylcarbonyl, alkylaminocarbonyl,heteroarylalkylaminocarbonyl, alkoxycarbonylalkylaminocarbonyl,acylaminoalkylaminocarbonyl, alkoxyaminocarbonyl,alkoxyalkylaminocarbonyl or alkylaminoalkylcarbonylamino, or R^(7tb) andR^(7tc) are linked to form a ring; and pharmaceutically acceptable saltsthereof.

In another embodiment, the invention pertains, at least in part, tosubstituted tetracycline compounds of Formula VIIIa:

wherein:

T* is N or CH;

R^(4*) is hydrogen or alkylamino.

R^(7ta*) is hydrogen or halogen;

R^(7tb*) is hydrogen, —CH═CHCN, hydroxyalkyl, —CONR^(7tba)R^(7tbb);—NHCOR^(7tbd);

R^(7tba) and R^(7tbb) are linked to form a ring; or R^(7tba) is hydrogenor alkyl and R^(7tbb) is hydrogen, alkoxy, alkyl or —(CH₂)_(x)R^(7tbc);

R^(7tbc) is amino, alkyl, alkoxycarbonyl, alkoxy, hydroxyl, aryl, aheterocyclic moiety or alkoxycarbonylamino;

R^(7tbd) is —(CH₂)_(y) R^(7tbe), wherein R^(7tbe) is amino;

R^(7tc*) is hydrogen, —O(CH₂)_(z)R^(tca); —CONHR^(tcb) or —NHCOR^(7td);wherein R^(tcb) is —(CH₂)_(w)R^(tcc) and R^(tca) and R^(tcc) are eachamino;

R^(7td) is alkoxy; or

R^(7tb*) and R^(7tc*) are linked to join a ring;

w, x, y and z are each, independently, an integer of between 0 and 5;and pharmaceutically acceptable salts thereof.

In one embodiment, the present invention also pertains to substitutedtetracycline compounds of Formula IX:

wherein:

R^(7u′) is hydrogen or cycloalkyl;

R^(7u″) is alkyl, alkylcarbonyloxyalkyloxycarbonyl, or aminoalkyl, orR^(7u′) and R^(7u″) are linked to form a ring; and pharmaceuticallyacceptable salts thereof.

In yet another embodiment, the invention pertains, at least in part, tosubstituted tetracycline compounds of Formula (IXa):

wherein

R^(7u*) is hydrogen;

R^(7u*′) is alkyl or —(CH₂)_(d)R^(7ua), wherein d is an integer frombetween 0 and 5 and R^(7ua) is amino; or

R^(7u*) and R^(7u*′) are linked to form a ring; and pharmaceuticallyacceptable salts thereof.

In another embodiment, the present invention also pertains, at least inpart, to substituted tetracycline compounds of Formula X:

wherein

R^(7v′) is alkyl, hydrogen or allyl;

R^(7v″) is arylalkyl or alkylcarbonyloxyalkyloxycarbonyl; or

R^(7v′) and R^(7v″) are linked to form a ring; and pharmaceuticallyacceptable salts thereof.

In yet another embodiment, the invention pertains, at least in part, tosubstituted tetracycline compounds of Formula Xa:

wherein

R^(7v*) is alkyl, hydrogen or allyl;

R^(7v*′) is arylalkyl or —COO(CH₂)_(f)R^(7va); or

R^(7v*) and R^(7v*′) are linked to form a ring;

f is an integer from between 0 and 5;

R^(7va) is alkylcarbonyloxy; and pharmaceutically acceptable saltsthereof.

In yet another embodiment, the present invention pertains, at least inpart, to a substituted tetracycline compound of the formula:

and pharmaceutically acceptable salts thereof.

In another embodiment, the invention pertains, at least in part tosubstituted tetracycline compounds of Formula XI:

wherein

R^(7w) is cycloalkyl;

R^(9W) is hydrogen or aminoalkyl, and pharmaceutically acceptable saltsthereof.

In yet another embodiment, the invention pertains, at least in part, tosubstituted tetracycline compounds of Formula XIa:

wherein

R^(7w*) is cycloalkyl;

R^(9w*) is hydrogen or CH₂NR^(9wa)R^(9wb);

R^(9wa) is alkyl and R^(9wb) is allyl; and pharmaceutically acceptablesalts thereof.

In one embodiment, the invention pertains, at least in part, tosubstituted tetracycline compounds of Formula XII:

wherein

R^(7x) is isopropyl, dimethylamino, or hydrogen;

R^(9x) is methyl, ethyl, furanyl, isopropyl, cyclopropyl,2-dimethyl-propyl, C(═O)NR^(9x′)R^(9x″), C(═O)OR^(9x′), C(═O)R^(9x′),thioazolyl, oxadiazolyl, hydrogen, phenyl, benzamidyl, dihydropyran,pyrazolyl, imidazolyl, or pyrrolyl;

R^(9x′) and R^(9x″) are each independently hydrogen, t-butyl, phenyl,hydroxyethyl, ethyl, 2-dimethylpropyl, or alkoxyethyl;

R^(10x) is hydrogen or alkyl; and pharmaceutically acceptable saltsthereof, provided that R^(7x) is not hydrogen or dimethylamino whenR^(9x) and R^(10x) are both hydrogen.

In one embodiment, the invention pertains, at least in part, tosubstituted tetracycline compounds of Formula XIII:

wherein

h is an integer from between 0 and 5;

R^(9*) is hydrogen, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, aminoalkyl, amido,arylalkenyl, arylalkynyl, thionitroso, alkylcarbonyl, arylcarbonyl,carboxylate, alkoxycarbonyl, aryloxycarbonyl or aminocarbonyl; andpharmaceutically acceptable salts thereof.

In yet another embodiment, the invention pertains, at least in part, tosubstituted tetracycline compounds of Formula XIV:

wherein

Z is —C═CH₂ or —CH₂;

R^(5y) is hydrogen or hydroxyl;

R^(7y) is hydrogen or dimethylamino;

R^(9y) is hydrogen;

R^(9y′) is —CH₂-cycloalkyl or —CH₂-substituted aryl; or

R^(9y) and R^(9y′) are linked to join a substituted piperidinyl ring ora tetracyclopyridinyl ring; and pharmaceutically acceptable saltsthereof.

In yet another embodiment, the invention pertains, at least in part, tosubstituted tetracycline compounds of Formula XV:

wherein

R^(9z) is hydrogen;

R^(9z′) is halogen substituted alkyl; or

R^(9z) and R^(9z′) are linked to form a substituted piperidinyl ring;and pharmaceutically acceptable salts thereof.

In yet another embodiment, the invention pertains, at least in part, tosubstituted tetracycline compounds of Formula XVI:

wherein

R^(9z*) is —(CH₂)_(t)R^(9za);

t is an integer from 0-1;

R^(9za) is hydrogen, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, aminoalkyl, amido,arylalkenyl, arylalkynyl, thionitroso, alkylcarbonyl, arylcarbonyl,carboxylate, alkoxycarbonyl, aryloxycarbonyl or aminocarbonyl; andpharmaceutically acceptable salts thereof.

In one embodiment, the invention pertains, at least in part, to apharmaceutical composition comprising a therapeutically effective amountof a tetracycline compound of the invention, e.g., a compound of FormulaI, II, III, IIIa, IV, IVa, V, Va, VI, VII, VIIa, VIII, VIIIa, IX, IXa,X, Xa, XI, XIa, XII, XIII, XIV, XV or XVI or a compound listed in Table2, and a pharmaceutically acceptable carrier.

In another further embodiment, the invention pertains, at least in part,to methods for treating subjects for tetracycline responsive states byadministering to them an effective amount of a tetracycline compound ofthe invention, e.g., a compound of Formula I, II, III, IIIa, TV, IVa, V,Va, VI, VII, VIIa, VIII, VIIIa, IX, IXa, X, Xa, XI, XIa, XII, XIII, XIV,XV or XVI or a compound listed in Table 2 or a tetracycline compoundotherwise described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical comparison of the modulation of carregeenaninduced inflammation in the rat paw edema model between doxycycline andcompound A.

FIG. 2 is a graphical comparison of the modulation of carregeenaninduced inflammation in the rat paw edema model between minocycline andcompound P.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains, at least in part, to novel substitutedtetracycline compounds. These tetracycline compounds can be used totreat numerous tetracycline compound-responsive states, such asbacterial infections, inflammation, and neoplasms, as well as otherknown applications for minocycline and tetracycline compounds ingeneral, such as blocking tetracycline efflux and modulation of geneexpression. The term “tetracycline compound” includes many compoundswith a similar ring structure to tetracycline. Examples of tetracyclinecompounds include: chlortetracycline, oxytetracycline, demeclocycline,methacycline, sancycline, chelocardin, rolitetracycline, lymecycline,apicycline; clomocycline, guamecycline, meglucycline, mepylcycline,penimepicycline, pipacycline, etamocycline, penimocycline, etc. Otherderivatives and analogues comprising a similar four ring structure arealso included (See Rogalski, “Chemical Modifications of Tetracyclines,”the entire contents of which are hereby incorporated herein byreference). Table 1 depicts tetracycline and several known othertetracycline derivatives.

TABLE 1

Other tetracycline compounds which may be modified using the methods ofthe invention include, but are not limited to,6-demethyl-6-deoxy-4-dedimethylaminotetracycline; tetracyclino-pyrazole;7-chloro-4-dedimethylaminotetracycline;4-hydroxy-4-dedimethylaminotetracycline;12α-deoxy-4-dedimethylaminotetracycline;5-hydroxy-6α-deoxy-4-dedimethylaminotetracycline;4-dedimethylamino-12α-deoxyanhydrotetracycline;7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline;tetracyclinonitrile; 4-oxo-4-dedimethylaminotetracycline 4,6-hemiketal;4-oxo-11a Cl-4-dedimethylaminotetracycline-4,6-hemiketal;5a,6-anhydro-4-hydrazon-4-dedimethyl amino tetracycline;4-hydroxyimino-4-dedimethylamino tetracyclines;4-hydroxyimino-4-dedimethylamino 5a,6-anhydrotetracyclines;4-amino-4-dedimethylamino-5a, 6 anhydrotetracycline;4-methylamino-4-dedimethylamino tetracycline;4-hydrazono-11a-chloro-6-deoxy-6-demethyl-6-methylene-4-dedimethylaminotetracycline; tetracycline quaternary ammonium compounds;anhydrotetracycline betaines; 4-hydroxy-6-methyl pretetramides; 4-ketotetracyclines; 5-keto tetracyclines; 5a, 11a dehydro tetracyclines; 11aCl-6, 12 hemiketal tetracyclines; 11a Cl-6-methylene tetracyclines; 6,13 diol tetracyclines; 6-benzylthiomethylene tetracyclines; 7,11a-dichloro-6-fluoro-methyl-6-deoxy tetracyclines; 6-fluoro(a)-6-demethyl-6-deoxy tetracyclines; 6-fluoro (β)-6-demethyl-6-deoxytetracyclines; 6-α acetoxy-6-demethyl tetracyclines; 6-βacetoxy-6-demethyl tetracyclines; 7, 13-epithiotetracyclines;oxytetracyclines; pyrazolotetracyclines; 11a halogens of tetracyclines;12a formyl and other esters of tetracyclines; 5, 12a esters oftetracyclines; 10, 12a-diesters of tetracyclines; isotetracycline;12-a-deoxyanhydro tetracyclines;6-demethyl-12a-deoxy-7-chloroanhydrotetracyclines; B-nortetracyclines;7-methoxy-6-demethyl-6-deoxyteiracyclines;6-demethyl-6-deoxy-5a-epitetracyclines; 8-hydroxy-6-demethyl-6-deoxytetracyclines; monardene; chromocycline; 5a methyl-6-demethyl-6-deoxytetracyclines; 6-oxa tetracyclines, and 6 thia tetracyclines.

In one embodiment, the invention pertains includes substitutedtetracycline compound of Formula I:

wherein

X is CHC(R¹³Y′Y), CR^(6′)R⁶, C═CR^(6′)R⁶, S, NR⁶, or O;

E is NR^(7d)R^(7e), OR^(7f) or (CH₂)₀₋₁C(═W′)WR^(7g);

W is O, S, NR^(7h) or CR^(7i)R^(7j);

W′ is O, S or NR^(7k);

R², R^(2′), R^(4′)R^(4a) and R^(4b) are each independently hydrogen,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety;

R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁴ is NR^(4a)R^(4b), alkyl, alkenyl, alkynyl, hydroxyl, halogen, orhydrogen;

R⁵ and R^(5′) are each hydroxyl, hydrogen, thiol, alkanoyl, aroyl,alkaroyl, aryl, heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkylcarbonyloxy, or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(7g), R^(7h), R^(7i),R^(7j) and R^(7k) are each independently hydrogen, allyl, alkyl,alkenyl, alkynyl, hydroxyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, amino, alkylamino, aminoalkyl, acyl, aryl, arylalkyl,alkylcarbonyloxy, alkylcarbonyloxylalkyl or arylcarbonyloxy, or R^(7c)and R^(7d) or R^(7e) and R^(7f) are linked to form a ring;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R⁹ is hydrogen, nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, aminoalkyl, amido, arylalkenyl, arylalkynyl, thionitroso, or (CH₂)₀₋₃(NR^(9c))₀₋₁C(═Z′)ZR^(9a);

Z is CR^(9d)R^(9e), S, NR^(9b) or O;

Z′ is O, S, or NR^(9f);

R^(9a), R^(9b), R^(9c), R^(9d), R^(9e) and R^(9f) are each independentlyhydrogen, acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,heteroaromatic or a prodrug moiety;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, amido, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts thereof.

In one embodiment, X is CR^(6′)R⁶, R⁴ is NR^(4a)R^(4b), R^(4a) andR^(4b) are each alkyl, and R², R^(2′), R³, R⁵, R^(5′) R⁶, R^(6′), R⁸,R⁹, R¹⁰,R¹¹ and R¹² are each hydrogen and R^(7a) and R^(7b) arehydrogen. In a further embodiment, E is OR^(7f), R^(7e) is hydrogen, andR^(7f) is alkyl (e.g., methyl, ethyl or t-butyl) or allyl.

In another embodiment, R^(7c) is alkyl (e.g., methyl or ethyl) andR^(7f) is alkyl (e.g., methyl, ethyl, isopropyl or t-butyl), which maybe substituted with a halogen (e.g., fluorine).

In yet another embodiment, R^(7e) and R^(7f) are linked to join a ring.

In one embodiment, E is NR^(7d)R^(7e), R^(7c) is alkyl (e.g., ethyl),R^(7d) is hydrogen and R^(7e) is alkyl (e.g., ethyl).

In a further embodiment, E is (CH₂)₀₋₁C(═W′)WR^(7g), such as E isC(═W′)WR^(7g). Accordingly, R^(7c) is alkyl (e.g., methyl), W and W′ areeach O and R^(7g) is alkyl (e.g., methyl). Alternatively, E isCH₂C(═W′)WR^(7g), R^(7c) is alkyl (e.g., methyl), W is CR^(7i)R^(7j) andR^(7g), R^(7i) and R^(7j) are each hydrogen. In a further embodiment, W′is NR^(7h) and R^(7h) is alkoxy (e.g., ethoxy).

In one embodiment, X is CR^(6′)R⁶, R⁴ is NR^(4a)R^(4b), R^(4a) andR^(4b) are each alkyl (e.g., methyl); R², R^(2′), R³, R^(4′) R⁵, R^(5′)R⁶, R^(6′), R^(7a), R^(7b), R^(7c), R⁸, R⁹, R¹⁰, R¹¹ and R¹² are eachhydrogen; E is OR^(7f); R^(7f) is allyl (e.g., CH₂═CHCH₂—) or alkyl(e.g., ethyl; isopropyl; t-butyl; alkoxy substituted alkyl (e.g.,methoxyethyl); halogen substituted alkyl (e.g., alkyl substituted withfluorine, for example, FCH₂CH₂—; F₂CHCH₂—; CF₃CH₂— or CF₂H—);alkylcarbonylalkyl (e.g., CH₃CO(CH₂)_(n)—, in which n is an integer from0-6, for example 1); alkoxycarbonylalkyl (e.g., CH₃OCO(CH₂)_(m)—, inwhich m is an integer from 0-6, for example 1) or carboxylatealkyl(HOOC(CH₂)_(q)—, in which q is an integer from 0-6, for example 1).

In one embodiment, X is CR^(6′)R⁶, R⁴ is NR^(4a)R^(4b), R^(4a) andR^(4b) are each alkyl (e.g., methyl); R², R^(2′), R³, R^(4′) R⁵, R^(5′)R⁶, R^(6′), R^(7a), R^(7b), R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each hydrogen;E is OR^(7f) and R^(7c) and R^(7f) are linked to join a ring, forexample, a 5- or 6-membered ring

In another embodiment, X is CR^(6′)R⁶, R⁴ is NR^(4a)R^(4b), R^(4a) andR^(4b) are each alkyl (e.g., methyl); R², R^(2′), R³, R^(4′) R⁵, R^(5′)R⁶, R^(6′), R^(7a), R^(7b)R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each hydrogen; Eis OR^(7f); R^(7c) and R^(7f) may be each independently alkyl (e.g.,methyl or ethyl).

In yet another embodiment, X is CR^(6′)R⁶, R⁴ is NR^(4a)R^(4b), R^(4a)and R^(4b) are each alkyl (e.g., methyl); R², R^(2′), R³, R^(4′) R⁵,R^(5′) R⁶, R^(6′), R^(7a), R^(7b), R^(7c), R⁸, R⁹, R¹⁰, R¹¹ and R¹² areeach hydrogen; E is NR^(7d)R^(7e); R^(7c) is alkyl (e.g., ethyl); R^(7d)is hydrogen and R^(7e) is alkyl (e.g., ethyl).

In another embodiment, X is CR^(6′)R⁶, R⁴ is NR^(4a)R^(4b), R^(4a) andR^(4b) are each alkyl (e.g., methyl); R², R^(2′), R³, R^(4′) R^(5′) R⁶,R^(6′), R^(7a), R^(7b), R^(7c), R⁸, R⁹, R¹⁰, R¹¹ and R¹² are eachhydrogen; E is —C(═W′)WR^(7g); W and W′ are each oxygen; R^(7c) is allyl(e.g., CH₂═CHCH₂—) and R^(7g) is alkoxy (e.g., methoxy).

In one embodiment, X is CR^(6′)R⁶, R⁴ is NR^(4a)R^(4b), R^(4a) andR^(4b) are each alkyl (e.g., methyl); R², R^(2′), R³, R^(4′) R⁵, R⁶,R^(6′), R^(7a), R^(7b), R^(7c), R⁸, R⁹, R¹⁰, R¹¹ and R¹² are eachhydrogen; E is —CH₂(C═W′)WR^(7g); R^(7c) is alkyl (e.g., methyl); W isCR^(7i)R^(7j); R^(7i), R^(7j) and R^(7g) are each hydrogen; W′ isNR^(7k) and R^(7k) is alkoxy (e.g., ethoxy).

In a further embodiment, In one embodiment, X is CR^(6′)R⁶, R⁴ isNR^(4a)R^(4b), R^(4a) and R^(4b) are each alkyl (e.g., methyl); R²,R^(2′), R³, R^(4′) R⁵, R^(5′) R⁶, R^(6′), R^(7a), R^(7b), R^(7c), R⁸,R⁹, R¹⁰, R¹¹ and R¹² are each hydrogen; E is —C(═W′)WR^(7g); W and W′are each oxygen; R^(7g) is alkylcarbonyloxyalkyl (e.g.,R^(7ga)R^(7gb)R^(7gC)COO(CH₂)_(r)— in which r is an integer between 1and 5 and R^(7ga)R^(7gb)R^(7gc) are each independently alkyl orhydrogen). In one embodiment, wherein R^(7ga)R^(7gb)R^(7gc) are eachalkyl (e.g., methyl); r is 1 and R^(7c) is hydrogen or alkyl (e.g.,cycloalkyl, for example, cyclopropyl).

Examples of substituted tetracycline compounds of Formula (I) include:

and pharmaceutically acceptable salts thereof.

In another embodiment, the invention includes substituted tetracyclinecompounds of Formula II:

wherein

X is CHC(R¹³Y′Y), CR^(6′)R⁶, C═CR^(6′)R⁶, S, NR⁶, or O;

J is NR^(7m)R^(7n), OR^(7o) or heteroaryl;

R², R^(2′), R^(4′), R^(4a) and R^(4b) are each independently hydrogen,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety;

R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a prodrug moiety;

R⁴ is NR^(4a)R^(4b), alkyl, alkenyl, alkynyl, hydroxyl, halogen, orhydrogen;

R⁵ and R^(5′) are each independently hydroxyl, hydrogen, thiol,alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, alkyl carbonyloxy, or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R^(7l), R^(7m), R^(7n) and R^(7o) are each independently hydrogen,alkyl, alkenyl, alkynyl, hydroxyl, alkoxy, amino, alkylamino,aminoalkyl, acyl, alkylthio, alkylsulfinyl, alkylsulfonyl, aryl,arylalkyl, alkylcarbonyloxy, or arylcarbonyloxy;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R⁹ is hydrogen, nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, aminoalkyl, amido, arylalkenyl, arylalkynyl, thionitroso or —(CH₂)₀₋₃(NR^(9c))₀₋₁C(═Z′)ZR^(9a);

Z is CR^(9d)R^(9e), S, NR^(9b) or O;

Z′ is O, S, or NR^(9f);

R^(9a), R^(9b), R^(9c), R^(9d), R^(9e) and R^(9f) are each independentlyhydrogen, acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,heteroaromatic or a prodrug moiety;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, amido, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts thereof.

In one embodiment, X is CR^(6′)R⁶, R⁴ is NR^(4a)R^(4b), R^(4a) andR^(4b) are each alkyl (e.g., methyl), R², R^(2′), R³, R^(4′), R⁵,R^(5′), R⁶, R^(6′), R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each hydrogen; J isOR^(7o); R^(7o) is alkyl (e.g., ethyl or t-butyl) and R^(7l) is alkyl(e.g., methyl) or aminoalkyl (e.g., dialkylaminoalkyl, such asdimethylaminoethyl).

In one embodiment, X is CR^(6′)R⁶, R⁴ is NR^(4a)R^(4b), R^(4a) andR^(4b) are each alkyl (e.g., methyl), R², R^(2′), R³, R^(4′), R⁵,R^(5′), R⁶, R^(6′), R⁸, R¹⁰, R¹¹ and R¹² are each hydrogen; R⁹ is amino;R^(7l) is alkyl (e.g., methyl); J is OR^(7o); R^(7o) is alkyl (e.g.,halogen substituted alkyl; such as fluorine substituted alkyl, forexample, CF₃CH₂—; ethyl or t-butyl).

In another embodiment, X is CR^(6′)R⁶, R⁴ is NR^(4a)R^(4b), R^(4a) andR^(4b) are each alkyl (e.g., methyl), R², R^(2′), R³, R^(4′), R⁵,R^(5′), R⁶, R^(6′), R⁸, R¹⁰, R¹¹ and R¹² are each hydrogen; R⁹ isaminoalkyl (e.g., t-butylaminomethyl); R^(7o) is alkyl (e.g., ethyl);and R^(7l) is alkyl (e.g., methyl).

In yet another embodiment, X is CR^(6′)R⁶, R⁴ is NR^(4a)R^(4b), R^(4a)and R^(4b) are each alkyl (e.g., methyl), R², R^(2′), R³, R^(4′), R⁵,R^(5′), R⁶, R^(6′), R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each hydrogen; J isNR^(7m)R^(7n); R^(7l) and R^(7m) are each hydrogen and R^(7n) is alkyl(e.g., t-butyl).

In a further embodiment, X is CR^(6′)R⁶, R⁴ is NR^(4a)R^(4b), R^(4a) andR^(4b) are each alkyl (e.g., methyl), R², R^(2′), R³, R^(4′), R⁵,R^(5′), R⁶, R^(6′), R^(7l), R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each hydrogen;J is heteroaryl (e.g., pyrrolyl).

In one embodiment, X is CR^(6′)R⁶, R⁴ is NR^(4a)R^(4b), R^(4a) andR^(4b) are each alkyl, and R², R^(2′), R³, R^(4a), R^(4b), R⁵, R^(5′)R⁶, R^(6′), R⁸, R¹⁰, R¹¹ and R¹² are each hydrogen, and J is OR^(7o).Accordingly, R⁹ is hydrogen and R^(7o) is alkyl (e.g., methyl, ethyl ort-butyl).

In another embodiment, R^(7l) is alkyl (e.g., methyl). Alternatively,R^(7l) is aminoalkyl, R⁹ is amino and R^(7o) is alkyl (e.g., ethyl ort-butyl).

In a further embodiment, R^(7l) is alkyl (e.g., methyl), R⁹ isaminoalkyl and R^(7o) is alkyl (e.g., ethyl). In yet another embodiment,R^(7l) is alkyl (e.g., methyl).

In one embodiment, J is NR^(7m)R^(7n), R^(9m), R^(7l) and R^(7m) areeach hydrogen and R^(7a) is alkyl (e.g., t-butyl).

In another embodiment, J is heteroaryl (e.g., pyrrolyl) and R⁹ andR^(7l) are each hydrogen.

Examples of substituted tetracycline compounds of Formula II include;

and pharmaceutically acceptable salts thereof.

In another embodiment, the invention pertains to substitutedtetracycline compounds of Formula III:

wherein

R^(7p) is acyl, alkylamino, or heteroaryl;

R^(10a) is hydrogen, aryl, carboxylate or alkoxycarbonyl;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; andpharmaceutically acceptable salts thereof.

In one embodiment, R^(7p) is alkylamino (e.g., dialkylamino, such asdimethylamino or substituted piperidinyl), acyl or heteroaryl (e.g.,pyrimidine or pyrazine).

In a further embodiment, R^(10a) is heteroaryl (e.g., oxazolyl),carboxylate or is alkoxycarbonyl (e.g., methoxycarbonyl).

In yet another embodiment, the invention includes substitutedtetracycline compounds of Formula IIIa:

wherein

R^(4p) is —N(CH₃)₂ or hydrogen;

R^(7p′) is hydrogen, amino, acyl, heteroaryl, aminoalkyl;

R^(10a′) is hydrogen, heteroaryl, alkoxycarbonyl, carboxylate, cyano,alkyl or alkoxy; and pharmaceutically acceptable salts thereof.

In one embodiment, R^(4p) is —N(CH₃)₂; R^(10a′) is hydrogen and R^(7p′)is acyl (e.g., CH₃CO—); heteroaryl (e.g., pyrimidinyl or pyrazinyl) oraminoalkyl (e.g., piperdinylalkyl, for example,4-methylpiperidinylmethyl)

In another embodiment, R^(4p) is —N(CH₃)₂; R^(7p) is dimethylamino andR^(10a′) is heteroaryl (e.g., oxazolyl or pyrazolyl), alkoxycarbonyl(e.g., methoxycarbonyl), carboxylate or cyano.

In yet another embodiment, R^(4p) is —N(CH₃)₂; R^(7p′) is hydrogen andR^(10a′) is alkoxycarbonyl (e.g., methoxycarbonyl) or alkyl (e.g.,methyl).

In another embodiment, R^(4p) is hydrogen; R^(7p′) is dimethylamino andR^(10a′) is alkyl (e.g., methyl) or alkoxy (e.g., methoxy).

Examples of substituted tetracycline compounds of Formulae III and IIIainclude:

and pharmaceutically acceptable salts thereof.

In another embodiment, the invention includes substituted tetracyclinecompounds of Formula IV:

wherein

L is O, NH or SH;

K is N or CR^(7p′);

M is N or CR^(7p″);

R^(7p′) is hydrogen;

R^(7p″) is hydrogen, aminoalkyl or alkoxycarbonylaminoalkyl, andpharmaceutically acceptable salts thereof.

In one embodiment, L is O. K is N, M is CR^(7p″) and R^(7p″) ishydrogen.

In another embodiment, L is O, K is CR^(7p′), R^(7p′) is hydrogen and Mis N. Alternatively, M is R^(7p″) and R^(7p″) is alkylamino (e.g., ismethylaminoalkyl, isopropylaminoalkyl or t-butylaminoalkyl) or isalkoxycarbonylaminoalkyl.

In another embodiment, L is SH, K is CR^(7p′),R^(7p′) is hydrogen and Mis N.

In yet another embodiment, L is NH, K is CR^(7p′) and M is CR^(7p″) andR^(7p′) and R^(7p″) are each hydrogen.

In another embodiment, includes substituted tetracycline compounds ofFormula IVa:

wherein

L* is O, NH or S;

*M is N, CH or CR^(7ps);

R^(7ps″) is aminoalkyl; and pharmaceutically acceptable salts thereof.

In one embodiment, *M is N and L* is S or O.

In another embodiment, *M is CH and L* is NH or O.

In a further embodiment, M is CR^(7ps″); R^(7ps″) is aminoalkyl (e.g.,—(CH₂)_(t)NR^(7pa)R^(7pb) in which t is an integer from 0 to 5 andR^(7pa) is hydrogen or alkyl and R^(7pb) is alkyl or alkoxycarbonyl). Inone embodiment, t is 1; R^(7pa) is alkyl (e.g., methyl) and R^(7pb) isalkyl (e.g., methyl) or alkoxycarbonyl (e.g., methoxycarbonyl). Inanother embodiment, R^(7pa) is hydrogen and R^(7pb) is alkyl (e.g.,methyl, isopropyl or t-butyl).

Examples of substituted tetracycline compounds of Formulae IV and IVainclude:

and pharmaceutically acceptable salts thereof.

The present invention also includes substituted tetracycline compoundsof Formula V:

wherein

R⁴ is hydrogen;

R⁴ is hydrogen or alkylamino;

R^(9f) is CR^(9g)NR^(9h) or CR^(9i)R^(9j)NR^(9k)R^(9l);

R^(9g), R^(9h), R^(9i), R^(9j), R^(9k) and R^(9l) are each independentlyhydrogen, alkyl, hydroxyl, amino, urea or alkoxy, or R^(9k) and R^(9l)are joined to form a ring; and pharmaceutically acceptable saltsthereof.

In one embodiment, R⁴ is hydrogen and R^(4′) is alkylamino (e.g.,dialkylamino, such as dimethylamino), R^(9k) is CR^(9g)NR^(9h), R^(9g)is hydrogen and R^(9h) is alkoxy (e.g., methoxy). In another embodiment,R^(9g) is alkyl (e.g., methyl) and R^(9h) is alkoxy (e.g., ethoxy).

In another embodiment, R^(9f) is CR^(9i)R^(9j)NR^(9k)R^(9l), R^(9i) andR^(9j) axe each hydrogen, R^(9k) is alkyl (e.g., methyl) and R^(9l) isalkoxy (e.g., methoxy or ethoxy). Alternatively, R^(9k) and R^(9l) arejoined to form a ring (e.g., a six-membered ring). In anotherembodiment, R^(9l) is amino, which may be substituted with analkylcarbonyl, or a urea moiety, which may be substituted with alkyl(e.g., t-butyl).

In yet another embodiment, R^(9k) is hydrogen, R^(9l) is alkoxy (e.g.,methoxy).

In a further embodiment, R⁴ and R^(4′) are each hydrogen, R^(9f) isCR^(9g)NR^(9h), R^(9g) is alkyl (e.g., methyl) and R^(9h) is alkoxy(e.g., methoxy).

In yet another embodiment, the invention includes substitutedtetracycline compounds of Formula Va:

wherein

Q is —CH₂ or —C═CH₂;

R⁴ is hydrogen;

R^(4′) is hydrogen or alkylamino;

R^(5a′) is hydrogen or hydroxyl;

R^(7qa) is —N(CH₃)₂ or hydrogen;

R^(9q) is CR^(9g′)NR^(9h′) or CR^(9i′)R^(9j′)NR^(9k′)R^(9l′);

R^(9g′), R^(9h′), R^(9i′), R^(9j′), R^(9k′) and R^(9l′) are eachindependently hydrogen, alkyl, hydroxyl, amino, urea or alkoxy, orR^(9k) and R^(9l) are joined to form a ring; and pharmaceuticallyacceptable salts thereof.

In one embodiment, Q is —C═CH₂; R^(4′) is alkylamino; R^(5a′) ishydroxyl and R^(7qa) is hydrogen and R^(9q) isCR^(9i′)R^(9j′)NR^(9k′)R^(9l′). In a further embodiment, R^(9i′) andR^(9j′) are each hydrogen and R^(9k′) is alkyl (e.g., methyl) andR^(9l′) is alkoxy (e.g., methoxy).

In another embodiment, Q is —CH₂; R^(4′) and R^(5a′) are each hydrogen;R^(7qa) is —N(CH₃)₂; R^(9q) is CR^(9g′)NR^(9h′); R^(9g′) is alkyl (e.g.,methyl) and R^(9h′) is alkoxy (e.g., methoxy).

In a further embodiment, Q is —CH₂; R^(4′) is aminoalkyl; R^(5a′) andR^(7qa) are each hydrogen; R^(9q) is CR^(9i′)R^(9j′)NR^(9k′)R^(9l′);R^(9i′), R^(9j′) and R^(9k′) are each hydrogen; R^(9l′) is alkoxy (e.g.,ethoxy).

In one embodiment, Q is —CH₂; R^(4′) is aminoalkyl; R^(5a′) and R^(7qa)are each hydrogen; R^(9q) is CR^(9i′)R^(9j′)NR^(9k′)R^(9l′); R^(9i′) andR^(9j′) are each hydrogen; R^(9k′) is alkyl (e.g., methyl) and whereinR^(9l′) is alkoxy (e.g., methoxy).

In a yet another embodiment, Q is —CH₂; R^(4′) is alkylamino, R^(5a′) ishydrogen; R^(7qa) is —N(CH₃)₂; R^(9q) is CR^(9g′)NR^(9h′); R^(9g′) ishydrogen and R^(9h′) is alkoxy (e.g., methoxy).

In a another embodiment, Q is —CH₂; R^(4′) is alkylamino, R^(5a′) ishydrogen; R^(7qa) is —N(CH₃)₂; R^(9q) is CR^(9g′)NR^(9h′); R^(9g′) isalkyl (e.g., methyl) and R^(9l′) is alkoxy (e.g., methoxy).

In a further embodiment, Q is —CH₂; R^(4′) is alkylamino, R^(5a′) ishydrogen; R^(7qa) is —N(CH₃)₂; R^(9q) is CR^(9i′)R^(9j′)NR^(9k′)R^(9l′);R^(9i′), R^(9j′) and R^(9k′) are each hydrogen and R^(9l′) is alkoxy(e.g., methoxy); alkylcarbonylamino (e.g., t-butylcarbonylamino) oralkylurea (e.g., t-butylurea).

In yet another embodiment, Q is —CH₂; R^(4′) is alkylamino, R^(5a′) ishydrogen; R^(7qa) is —N(CH₃)₂; R^(9q) is CR^(9i′)R^(9j′)NR^(9k′)R^(9l′);R^(9i′) and R^(9j′) are each hydrogen; R^(9k′) is alkyl (e.g., methyl)and R^(9l′) is alkoxy (e.g., methoxy).

Examples of substituted tetracycline compounds of Formulae V and Vainclude:

and pharmaceutically acceptable salts thereof.

In one embodiment, the invention also includes substituted tetracyclinecompounds of Formula VI;

wherein

X is CHC(R¹³Y′Y), CR^(6′)R⁶, C═CR^(6′)R⁶, S, NR⁶, or O;

p is a single bond or a double bond;

Q is CR^(7s) when p is a double bond or Q is CR^(7s′)R^(7s″) when p is asingle bond;

R², R^(2′), R^(4′), R^(4a) and R^(4b) are each independently hydrogen,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety;

R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a prodrug moiety;

R⁴ is NR^(4a)R^(4b), alkyl, alkenyl, alkynyl, hydroxyl, halogen, orhydrogen;

R⁵ and R^(5′) are each independently hydroxyl, hydrogen, thiol,alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, alkyl carbonyloxy, or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R^(7s), R^(7s′) and R^(7s″) are each hydrogen, alkyl, alkenyl, alkynyl,hydroxyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, amino,aminoalkyl, alkylamino, aryl, acyl, arylalkyl, alkyl carbonyloxy, orarylcarbonyloxy;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R⁹ is hydrogen, nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, aminoalkyl, amido,arylalkenyl, arylalkynyl, thionitroso or—(CH₂)₀₋₃(NR^(9c))₀₋₁C(═Z′)ZR^(9a);

Z is CR^(6d)R^(9e), S, NR^(9b) or O;

Z′ is O, S, or NR^(9f);

R^(9a), R^(9b), R^(9c), R^(9d), R^(9e) and R^(9f) are each independentlyhydrogen, acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,heteroaromatic or a prodrug moiety;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, amido, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts thereof.

In one embodiment, X is CR^(6′)R⁶, R⁴ is NR^(4a)R^(4b), R^(4a) andR^(4b) are each alkyl (e.g., methyl) and R², R^(2′), R³, R⁴, R⁵, R^(5′),R⁶, R^(6′), R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each hydrogen. In anotherembodiment, p is a double bond and Q is CR^(7s). In a furtherembodiment, R^(7s) is amino, alkylamino (e.g., methylamino) ordialkylamino (e.g., dimethylamino).

Examples of substituted tetracycline compounds of Formula VI include:

and pharmaceutically acceptable salts thereof.

In one embodiment, the substituted tetracycline compounds of theinvention include compounds of Formula VII:

wherein

X is CR^(6′)R⁶;

R⁵ is hydroxyl or hydrogen;

R^(5′) is hydrogen;

R^(6′) hydrogen or alkyl;

R⁶ is hydrogen;

R^(7r) is hydrogen or alkylamino;

R^(9m) is heteroaryl, aminocarbonyl, hydroxyaminocarbonyl,alkoxyaminocarbonyl or —CR^(9m) NR^(9m″);

R^(9m′) and R^(9m″) are each hydrogen, hydroxy, alkyl, alkenyl, alkynyl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl; and pharmaceutically acceptable salts thereof.

In one embodiment, X is CR^(6′)R⁶; and R⁵, R^(5′), R⁶ and R^(6′) areeach hydrogen, R⁷ is alkylamino (e.g., dialkylamino such asdimethylamino) and R^(9m) is heteroaryl (e.g., oxazolyl, isoxazolyl,pyrazolyl or pyridinyl), aminocarbonyl (e.g., dialkylaminocarbonyl, suchas diethylaminocarbonyl, dimethylaminocarbonyl,propylmethylaminocarbonyl, pyrrolidinyl or piperidinyl),hydroxyaminocarbonyl (e.g., hydroxyaminomethylcarbonyl),alkoxyaminocarbonyl (e.g., methoxyaminocarbonyl), alkoxycarbonyl (e.g.,ethoxycarbonyl or methoxycarbonyl) or —CR^(9m′)NR^(9m″). Accordingly,R^(9m′) is alkyl (e.g., methyl) and R^(9m″) is alkoxy (e.g., methoxy).

In another embodiment, X is CR^(6′)R⁶; R⁶ is alkyl, R⁵ is hydroxyl andR^(5′) and R^(6′) are each hydrogen and R^(9m) is heteroaryl.

In yet another embodiment, the substituted tetracycline compounds of theinvention include compounds of Formula VIIa:

wherein

X is CR^(6*′)R^(6*);

R^(5*) is hydrogen;

R^(5*′) is hydrogen hydroxyl;

R^(6*′) is hydrogen or alkyl;

R^(6*) is hydrogen;

R^(7r*) is hydrogen, alkyl, heteroaryl, acyl or alkylamino;

R^(9m*) is aminoalkyl, a heterocyclic moiety, aryl, —CONR^(9ma)R^(9mb);—COR^(9m*′), —COOR^(9m*″), alkyl, cycloalkyl or hydrogen;

R^(9m*′) is aminoalkyl, aryl or alkyl;

R^(9m*″) is alkyl, alkoxyalkyl or hydroxyalkyl;

R^(9ma) and R^(9mb) are each hydrogen, hydroxyl, alkyl, hydroxyalkyl,aryl or alkoxy or are linked to form a ring; and pharmaceuticallyacceptable salts thereof.

In one embodiment, R^(5*′) is hydroxyl; R^(6*′) is alkyl; R^(7r*) ishydrogen; R^(9m*) is aryl (e.g., heteroaryl, such as oxazolyl).

In yet another embodiment, R^(5*′) and R^(6*′) are each hydrogen;R^(7r*) is alkylamino (e.g., dialkylamino, for example, dimethylamino);and R^(9m*) is aminoalkyl (e.g., dimethylaminopropyl); a heterocyclicmoiety (e.g., a dihydropyranyl moiety, for example

aryl (e.g., phenyl or benzamido; or a heteroaryl moiety such asoxazolyl, oxadiazolyl (e.g., alkyl substituted oxadiazolyl, such asmethyl or isopropyl substituted oxazolyl), isoxazolyl, pyrazolyl,pyridinyl, thiazolyl, methylpyrazolyl, methylimidazolyl, oxadiazolyl,furanyl (e.g., carboxylate substituted furanyl) or pyrrolyl); alkyl(e.g., methyl, ethyl, isopropyl, neopentyl, or trifluoromethyl) orcycloalkyl (e.g., cyclopropyl).

In yet another embodiment, R^(5*′) and R^(6*′) are each hydrogen;R^(7r*) is alkylamino (e.g., dialkylamino, for example, dimethylamino);and R^(9m*) is —CONR^(9ma)R^(9mb-); R^(9ma) is hydrogen and R^(9mb) ishydroxyl, hydroxyalkyl (e.g., hydroxyethyl), alkoxy (e.g., methoxy),aryl (e.g., phenyl) or alkyl (e.g., t-butyl). In a further embodiment,R^(9ma) is alkyl (e.g., methyl, ethyl or propyl) and R^(9mb) is alkyl(e.g., methyl, ethyl or propyl) or hydroxyl. In yet another embodiment,R^(9ma) and R^(9mb) are linked to form a ring (e.g., a 5- or 6-memberedring).

In one embodiment, R^(5*′) and R^(6*′) are each hydrogen; R^(7r*) isalkylamino (e.g., dialkylamino, for example, dimethylamino); and R^(9m*)is —COOR^(9m*″) and R^(9m*″) is alkyl (e.g., methyl, ethyl orneopentyl), alkoxyalkyl (e.g., methoxyethyl) or hydroxyalkyl (e.g.,hydroxyethyl).

In another embodiment, R^(5′) and R^(6*′) are each hydrogen; R^(7r*) isalkylamino (e.g., dialkylamino, for example, dimethylamino); R^(9m*) is—COR^(9m*′) and R^(9m*′) is alkyl (e.g., ethyl or neopentyl), aminoalkyl(e.g., dimethylaminoethyl) or phenyl.

In one more embodiment, R^(5*′) and R^(6*′) are each hydrogen; R^(9m*)is hydrogen and R^(7r*) is alkyl (e.g., methyl or isopropyl), aryl(e.g., heteroaryl, for example oxazolyl) or acyl.

Examples of substituted tetracycline compounds of Formulae VII and VIIainclude:

and pharmaceutically acceptable salts thereof.

In yet another embodiment, the substituted tetracycline compoundsinclude compounds of Formula VIII:

wherein:

T is N or CR^(7td);

R⁴ is alkylamino or hydrogen;

R^(4′) is hydrogen;

R^(7te) is hydrogen.

R^(7ta), R^(7tb), R^(7tc) and R^(7td) are each independently hydrogen,halogen, hydroxyalkyl, hydroxyalkylaminocarbonyl, alkylaminoalkyloxy,aminocarbonyl, alkylaminoalkylaminocarbonyl, aminoalkylaminocarbonyl,methylpiperazinylcarbonyl, alkylaminocarbonyl,heteroarylalkylaminocarbonyl, alkoxycarbonylalkylaminocarbonyl, acylaminoalkylaminocarbonyl, alkoxyaminocarbonyl, alkoxyalkylaminocarbonylor alkylaminoalkylcarbonylamino, or R^(7tb) and R^(7tc) are linked toform a ring; and pharmaceutically acceptable salts thereof.

In one embodiment, R⁴ is alkylamino (e.g., dialkylamino such asdimethylamino), R^(4′) is hydrogen, T is N and R^(7ta) and R^(7td) areeach hydrogen and R^(7tb) and R^(7tc) are linked to form a ring. Inanother embodiment, R^(7tc) is alkylaminoalkyloxy (e.g.,dialkylaminoalkyloxy such as dimethylaminoalkyloxy).

In a further embodiment, N is CR^(7td) and R^(7ta), R^(7tc) and R^(7td)are each hydrogen. In another embodiment, R^(7td) is hydroxyalkyl,aminocarbonyl, alkylaminoalkylaminocarbonyl (e.g.,methylaminoalkylaminocarbonyl), dialkylaminoalkylaminocarbonyl (e.g.,dimethylaminoalkylaminocarbonyl, diethylaminoalkylaminocarbonyl,diisopiopylaminoalkylaminocarbonyl, pyrrolidinylalkylaminocarbonyl orpiperidinylalkylaminocarbonyl), hydroxyalkylaminocarbonyl,aminoalkylaminocarbonyl, methylpiperazinylcarbonyl,Alkylaminoalkylaminocarbonyl, heteroarylalkylaminocarbonyl (e.g.,furanylalkylaminocarbonyl), alkoxycarbonyl alkyl aminocarbonyl (e.g.,ethoxycarbonylalkylaminocarbonyl), acylaminoalkylaminocarbonyl,alkoxyaminocarbonyl (e.g., methoxyaminocarbonyl),alkoxyalkylaminocarbonyl (e.g., methoxyalkylaminocarbonyl) oralkylaminoalkylcarbonylamino (e.g., dialkylaminoalkylcarbonylamino suchas dimethylaminoalkylcarbonylamino).

In another embodiment, R^(7ta), R^(7tb) and R^(7td) are hydrogen andR^(7tc) is alkylaminocarbonyl.

In yet another embodiment, R^(7ta) is halogen (e.g., fluorine), R^(7tc)and R^(7td) are each hydrogen and R^(7tb) isalkylaminoalkylaminocarbonyl (e.g., dialkylaminoalkylaminocarbonyl suchas dimethylaminoalkylaminocarbonyl).

In one embodiment, R⁴ and R^(4′) are each hydrogen, R^(7ta), R^(7tc) andR^(7td) are each hydrogen and R^(7tb) is alkylaminoalkylaminocarbonyl(e.g., dialkylaminoalkylaminocarbonyl such asdimethylaminoalkylaminocarbonyl).

In a further embodiment, the substituted tetracycline compounds of theinvention include compounds of Formula VIIIa:

wherein:

T* is N or CH;

R^(4*) is hydrogen or alkylamino.

R^(7ta*) is hydrogen or halogen;

R^(7tb*) is hydrogen, —CH═CHCN, hydroxyalkyl, —CONR^(7tba)R^(7tbb);—NHCOR^(7tbd);

R^(7tba) and R^(7tbb) are linked to form a ring; or R^(7tba) is hydrogenor alkyl and R^(7tbb) is hydrogen, alkoxy, alkyl or —(CH₂)_(x)R^(7tbc);

R^(7tbc) is amino, alkyl, alkoxycarbonyl, alkoxy, hydroxyl, aryl, aheterocyclic moiety or alkoxycarbonylamino;

R^(7tbd) is (CH₂)_(y) R^(7tbc), wherein R^(7tbe) is amino;

R^(7tc*) is hydrogen, —O(CH₂)_(z)R^(tca); —CONHR^(tcb) or —NHCOR^(7td);wherein R^(tcb) is —(CH₂)_(w)R^(tcc) and R^(tca) and R^(tcc) are eachamino;

R^(7td) is alkoxy; or

R^(7tb*) and R^(7tc*) are linked to join a ring;

w, x, y and z are each, independently, an integer of between 0 and 5;and pharmaceutically acceptable salts thereof.

In one embodiment, T* is CH and R^(4*) is alkylamino (e.g.,dimethyamino); R^(7ta*) and R^(7tc*) are each hydrogen and R^(7tb*) is—CH═CHCN, hydroxyalkyl (e.g., hydroxypropyl) or —CONR^(7tba)R^(7tbb). Inone embodiment, R^(7tba) is hydrogen and R^(7tbb) is hydrogen, alkoxy(e.g., methoxy), alkyl (e.g., butyl) or R^(7tbb) is —(CH₂)_(x)R^(7tbc),in which, when x is 1, R^(7tbc) may be aryl (e.g., heteroaryl, such asfuranyl); when x is 2, R^(7tbe) may be amino (e.g., —NH₂, methylamino,dimethylamino, diethyl amino or diisopropylamino), alkoxycarbonyl (e.g.,ethoxycarbonyl), hydroxyl, alkoxy (e.g., methoxy), alkylcarbonylamino(e.g., methylcarbonylamino) or a heterocyclic moiety (e.g., piperidinylor morpholinyl); when x is 3, R^(7tbc) may be amino (e.g.,dimethylamino) or alkoxy (e.g., methoxy) or when x is 4, R^(7tbc) may beamino (e.g., dimethylamino).

In another embodiment, R^(7tba) is alkyl (e.g., methyl) and R^(7tbb) is—(CH₂)_(x)R^(7tbc) in which, when x is 2, R^(7tbc) may be amino (e.g.,dimethylamino).

In a further embodiment, R^(7tba) and R^(7tbb) are linked to join a ring(e.g., a 5- or 6-membered ring, for example,

In one embodiment, T* is CH and R^(4*) is alkylamino (e.g.,dimethyamino); R^(7ta*) and R^(7tc*) are each hydrogen and R^(7tb*) is—NHCOR^(7tbd); R^(7tbd) is —(CH₂)_(y)R^(7tbc), y is 2 or 4 and R^(7tbc)is amino (e.g., dimethylamino).

In another embodiment, T* is CH and R^(4*) is alkylamino (e.g.,dimethyamino); R^(7tc*) is hydrogen; R^(7ta*) is a halogen (e.g.,fluorine); R^(7tb*) is —CONR^(7tba)R^(7tbb) in which R^(7tba) ishydrogen and R^(7tbb) is (CH₂)_(x)R^(7tbc). In one embodiment, x is 2and R^(7tbc) is amino (e.g., dimethylamino).

In a further embodiment, T* is CH and R^(4*) is hydrogen; R^(7ta*) andR^(7tc*) are each hydrogen; R^(7tb*) is —CONR^(7tba)R^(7tbb); R^(7tba)is hydrogen; R^(7tbb) is —(CH₂)_(x)R^(7tbc) in which when x is 2,R^(7tbc) may be amino (e.g., dimethylamino).

In yet another embodiment, T* is CH and R^(4*) is alkylamino (e.g.,dimethyamino); R^(7ta*) and R^(7tb*) are each hydrogen; R^(7tc*) is—CONHR^(tcb); R^(tcb) is —(CH₂)_(w)R^(tcc) in which when w is 2,R^(7tc*) may be —NHCOR^(7td) and R^(7td) is alkoxy (e.g., halogensubstituted alkoxy, for example, is —CH₂CH₂F).

In another embodiment, T* is N and R^(4*) is alkylamino (e.g.,dimethylamino); R^(7ta*) is hydrogen and R^(7b*) and R^(7c*) are linkedto form a ring (e.g.,

In one embodiment, T* is N and R^(4*) is alkylamino (e.g.,dimethylamino); R^(7ta*) and R^(7tb*) are each hydrogen and R^(7tc*) is—O(CH₂)_(z)R^(tca) in which z is 3.

Examples of substituted tetracycline compounds of Formulae VIII andVIIIa include:

and pharmaceutically acceptable salts thereof.

The substituted tetracycline compounds of the invention also includecompounds of Formula IX:

wherein:

R^(7u′) is hydrogen or cycloalkyl;

R^(7u″) is alkyl, alkylcarbonyloxyalkyloxycarbonyl, or aminoalkyl, orR^(7u′) and R^(7u″) are linked to form a ring; and pharmaceuticallyacceptable salts thereof.

In one embodiment, R^(7u′) is hydrogen and R^(7u″) is alkyl (e.g.,t-butyl, isopropyl, cyclopropyl or cyclopentyl). Alternatively, R^(7u″)is alkylcarbonyloxyalkyloxycarbonyl or aminoalkyl (e.g.,dialkylaminoalkyl, such as dimethylaminoalkyl).

In another embodiment, R^(7u′) and R^(7u″) are linked to form a ring(e.g., a substituted piperidinyl ring). Examples of substituents on thering include, for example, an alkyl substituent, which may or may not besubstituted with one or more halogens (e.g., fluorine). In oneembodiment, the ring is an indole ring.

In another embodiment, R^(7u′) is cycloalkyl (e.g., cyclopropyl) andR^(7u″) is alkylcarbonyloxyalkyloxycarbonyl.

In another embodiment, the substituted tetracycline compounds of theinvention include compounds of Formula IXa:

wherein:

R^(7u*) is hydrogen;

R^(7u*′) is alkyl or (CH₂)_(d)R^(7ua), wherein d is an integer frombetween 0 and 5 and R^(7ua) is amino; or

R^(7u*) and R^(7u*′) are linked to form a ring; and pharmaceuticallyacceptable salts thereof.

In one embodiment, R^(7u*) is hydrogen and R^(7u*′) is alkyl (e.g., acycloalkyl moiety, such as is cyclopropyl or cyclopentyl, or t-butyl orisopropyl)

In another embodiment, R^(7u*) is hydrogen and R^(7u*′) is—(CH₂)_(d)R^(7ua), in which d may be 4 and R^(7ua) may be amino (e.g.,dimethylamino).

In a further embodiment, R^(7u*) and R^(7u*′) are linked to form a ring(e.g., a 5- or 6-membered ring, such as

Examples of substituted tetracycline compounds of Formulae IX or IXainclude:

and pharmaceutically acceptable salts thereof.

The substituted tetracycline compounds of the invention includecompounds of Formula X:

wherein

R^(7v′) is alkyl, hydrogen or allyl;

R^(7v″) is arylalkyl or alkylcarbonyloxyalkyloxycarbonyl; or

R^(7v′) and R^(7v″) are linked to form a ring; and pharmaceuticallyacceptable salts thereof.

In one embodiment, R^(7v′) is alkyl or allyl and R^(7v″) isalkylcarbonyloxyalkyloxycarbonyl.

In another embodiment, R^(7v′) and R^(7v″) are linked to form a ring.

In yet another embodiment, R^(7v′) is hydrogen and R^(7v″) is arylalky,such as for example, phenylalkyl, which may be substituted with one ormore halogens (e.g., fluorine).

In one embodiment, the substituted tetracycline compounds of theinvention include compounds of Formula Xa:

wherein

R^(7v*) is alkyl, hydrogen or allyl;

R^(7v*′) is arylalkyl or —COO(CH₂)_(f)R^(7va); or

R^(7v*) and R^(7v*′) are linked to form a ring;

f is an integer from between 0 and 5;

R^(7va) is alkylcarbonyloxy; and pharmaceutically acceptable saltsthereof.

In one embodiment, R^(7v*) is hydrogen and R^(7v*′) is arylalkyl (e.g.,benzyl, for example, 2,6-difluorobenzyl).

In another embodiment, R^(7v*) is alkyl (e.g., methyl) or allyl andR^(7v*′) is —COO(CH₂)_(f)R^(7va), in which f may be 1 and R^(7va) may bet-butylcarbonyloxy.

In a further embodiment, R^(7v*) and R^(7v*′) are linked to form a ring(e.g., a 5- or 6-membered aliphatic or aromatic ring, for example,

Examples of substituted tetracycline compounds of Formulae X and Xainclude:

and pharmaceutically acceptable salts thereof.

In yet another embodiment, the substituted tetracycline compoundsinclude compounds of the formula:

and pharmaceutically acceptable salts thereof.

In another embodiment, the substituted tetracycline compounds of theinvention include compounds of Formula XI:

wherein

R^(7w) is cycloalkyl;

R^(9w) is hydrogen or aminoalkyl; and pharmaceutically acceptable saltsthereof.

In one embodiment, the cycloalkyl is cyclopropyl. In another embodiment,R^(9w) is hydrogen. In a further embodiment, R^(9w) is aminoalkyl (e.g.,dialkylamino).

In yet another embodiment, the substituted tetracycline compounds of theinvention include compounds of Formula XIa:

wherein

R^(7w*) is cycloalkyl;

R^(9w*) is hydrogen or —CH₂NR^(9wa)R^(9wb);

R^(9wa) is alkyl and R^(9wb) is allyl; and pharmaceutically acceptablesalts thereof.

In one embodiment, the cycloalkyl is cyclopropyl and R^(9w*) ishydrogen. In another embodiment, R^(9w*) is —CH₂NR^(9wa)R^(9wb), inwhich R^(9wa) is methyl.

Examples of substituted tetracycline compound of Formulae XI and XIainclude:

and pharmaceutically acceptable salts thereof.

In another embodiment, the tetracycline compounds of the inventioninclude substituted tetracycline compounds of formula XII:

wherein

R^(7X) is isopropyl, dimethylamino, or hydrogen;

R^(9x) is methyl, ethyl, furanyl, isopropyl, cyclopropyl,2-dimethyl-propyl, C(═O)NR^(9x′)R^(9x″), C(═O)OR^(9x′), C(═O)R^(9x′),thioazolyl, oxadiazolyl, hydrogen, phenyl, benzamidyl, dihydropyran,pyrazolyl, imidazolyl, or pyrrolyl;

R^(9x′) and R^(9x″) are each independently hydrogen, t-butyl, phenyl,hydroxyethyl, ethyl, 2-dimethylpropyl, or alkoxyethyl;

R^(10x) is hydrogen or alkyl; and pharmaceutically acceptable saltsthereof, provided that R^(7x) is not hydrogen or dimethylamino whenR^(9x) and R^(10x) are both hydrogen.

In a further embodiment, R^(7x) is isopropyl and R^(9x) and R^(10x) areeach hydrogen.

In another embodiment, R^(7x) is dimethylamino. Examples of R^(9x)groups include: ethyl, methyl, isopropyl, cyclopropyl, 2-dimethylpropyl,phenyl, 4-benzamidyl, 2-furanyl, 3,4-dihydropyranyl, and 2-thioazolyl.In certain cases, the R^(9x) groups can be further substituted. Forexample, R^(9x) furanyl groups may be substituted with substituents suchas carboxylate (—COOH).

In another embodiment, R^(9x) is C(═O)NR^(9x′)R^(9x″) and R^(9x′) ishydrogen. Examples of R^(9x″) include phenyl and t-butyl. Other optionsfor R^(9x) include C(═O)OR^(9x′), wherein R^(9*) may be 2-hydroxyethyl,2-dimethylpropyl or 2-methyoxyethyl. R^(9x) also may be C(═O)R^(9x′),when R^(9x′) is ethyl or 2-dimethylpropyl.

In another embodiment, R^(9x) is substituted oxadiazolyl. Examples ofsubstituents include those which allow the compound of the invention toperform its function, such as, but not limited to alkyl, e.g., methyl orisopropyl.

In yet another embodiment, R^(9x) is substituted pyrazolyl, imidazolyl,or pyrrolyl. R^(9x) may be substituted with one or more substituents.Examples of such substituents include alkyl, e.g., methyl, ethyl, etc.In another further embodiment, the pyrazolyl, imidazolyl, and/orpyrrolyl groups are N-substituted, e.g., N-methyl substituted.

In another further embodiment, R^(9x) is hydrogen and R^(10x) is methyl.

In another embodiment, the substituted tetracycline compounds of theinvention include compounds of Formula XIII:

wherein

h is an integer from between 0 and 5;

R^(9*) is hydrogen, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, aminoalkyl, amido,arylalkenyl, arylalkynyl, thionitroso, alkylcarbonyl, arylcarbonyl,carboxylate, alkoxycarbonyl, aryloxycarbonyl or aminocarbonyl; andpharmaceutically acceptable salts thereof.

In one embodiment, h is 2 or 3 and the amino is alkylamino (e.g.,dimethylamino).

Examples of substituted tetracycline compounds of Formula XIII include:

and pharmaceutically acceptable salts thereof.

The substituted tetracyclines of the invention also include compounds ofFormula XIV:

wherein

Z is —C═CH₂ or —CH₂;

R^(5y) is hydrogen or hydroxyl;

R^(7y) is hydrogen or dimethylamino;

R^(9y) is hydrogen;

R^(9y′) is —CH₂-cycloalkyl or —CH₂— substituted aryl; or

R^(9y) and R^(9y′) are linked to join a substituted piperidinyl ring ora tetracyclopyridinyl ring; and pharmaceutically acceptable saltsthereof.

In one embodiment, Z is —C═CH₂, R^(5y) is hydroxyl, R^(7y) is hydrogenand R^(9y) and R^(9y′) are linked to join a substituted piperidinyl ring(e.g., a halogen substituted piperidinyl ring, such as a fluorinesubstituted piperidinyl ring, for example,

In another embodiment, Z is CH₂, R^(5y) and R^(7y) are each hydrogen andR^(9y) and R^(9y′) are linked to join a substituted piperidinyl ring(e.g., a halogen substituted piperidinyl ring, such as a fluorinesubstituted piperidinyl ring, for example,

In yet another embodiment, Z is CH₂, R^(5y) is hydrogen; R^(7y) isdimethylamino; R^(9y) is hydrogen and R^(9y′) is —CH₂-cycloalkyl (e.g.,—CH₂-cyclopropyl); —CH₂-substituted aryl (e.g., hydroxyl substitutedphenyl such as 2,3-diphenolyl); or R^(9y) and R^(9y′) are linked to forma substituted piperidinyl ring (e.g.,

or a tetrahydropyridinyl ring (e.g.,

Examples of substituted tetracycline compounds of Formula XIV include:

and pharmaceutically acceptable salts thereof.

The substituted tetracycline compounds of the invention also includecompounds of Formula XV:

wherein

R^(9z) is hydrogen;

R^(9z′) is halogen substituted alkyl; or

R^(9z) and R^(9z′) are linked to form a substituted piperidinyl ring;and pharmaceutically acceptable salts thereof.

In one embodiment, R^(9z) is hydrogen and R^(9z′) is halogen substitutedalkyl, such as CF₃(CH₂)_(p)—, wherein p is an integer from 0 to 5 (e.g.,1).

In another embodiment, R^(9z) and R^(9z′) are linked to form asubstituted piperidinyl ring (e.g., an alkyl substituted piperidinylring, such as, methyl substituted piperidinyl or trifluoromethylsubstituted piperidinyl, for example,

Examples of substituted tetracycline compounds of Formula XV include:

and pharmaceutically acceptable salts thereof.

In another embodiment, the substituted tetracycline compounds includecompounds of Formula XVI:

wherein

R^(9z*) is —(CH₂)_(t)R^(9za);

t is an integer from 0-1;

R^(9za) is hydrogen, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, aminoalkyl, amido, arylalkenyl, arylalkynyl, thionitroso, alkylcarbonyl, arylcarbonyl,carboxylate, alkoxycarbonyl, aryloxycarbonyl or aminocarbonyl; andpharmaceutically acceptable salts thereof.

In one embodiment, t is 1 and R^(9za) is amino (e.g., dimethylamino ortrimethylammonium).

Examples of substituted tetracycline compounds of Formula XVI include:

and pharmaceutically acceptable salts thereof.

Methods for Synthesizing Tetracycline Compounds of the Invention

The substituted tetracycline compounds of the invention can besynthesized using the methods described in the following schemes and byusing art recognized techniques. All novel substituted tetracyclinecompounds described herein are included in the invention as compounds.

In Scheme 1, a general synthetic scheme for synthesizing 7-substitutedIn Scheme 1, a general synthetic scheme for synthesizing 7-substitutedtetracyclines is shown. A palladium catalyzed coupling of aniodosancycline (1) is performed to form a 7-substituted aldehydeintermediate (2). The aldehyde intermediate is reduced in the presenceof a hydroxylamine to give the desired product (3). Compounds P, Y, U,DR and DS may be synthesized as described in Scheme 1.

7- and 9-substituted tetracycline compounds may be synthesized byreacting the 7-iodo-9-aminoalkyl sancycline derivative (4) withtrimethylsilylethyne in the presence of a palladium catalyst to yield a7-substituted alkynyl intermediate. Subsequent acid hydrolysis yieldsthe 7-acyl intermediate (5). Further derivitization of the 9-positionmay be accomplished by reductive alkylation of the amino group witht-butyl aldehyde, hydrogen and palladium on carbon to form compound 6,which can then be reacted with a primary hydroxyl amine to form theoxime 7. Compound N may be synthesized as described in Scheme 2.

7- and 9-substituted tetracycline compounds may also be prepared asshown in Scheme 3. Beginning with a 7-iodo-9-nitro substitutedsancycline derivative (8), a Hiyama coupling followed by acid hydrolysisyields a 7-acyl-9-nitro intermediate (9). The nitro moiety may then bereduced to the amino group by hydrogen gas in the presence of apalladium catalyst (10). Reaction of the acyl group with a primaryhydroxylamine provides the product 11. Compounds M, Q, R, DT and DU maybe synthesized as shown in Scheme 3.

Scheme 4 also provides a method for synthesizing 7-substitutedtetracyclines. As described above, a palladium catalyzed carbonylationof an iodosancycline (1) is performed to form a 7-substituted aldehydeintermediate (2). The aldehyde intermediate is reduced in the presenceof a hydroxylamine to give compound 12, which may then be reacted withformaldehyde and triethylamine, followed by reduction to give thedesired product (3). Compounds AA, AM, AB, AE, AF, AG, DV, DW, DX, DY,DZ, EA, EB, EC, ED, EE, EF, EG and EH may be synthesized as illustratedin Scheme 4.

Scheme 5 details the synthesis of substituted tetracyclines with hydroxyin the 10-position. A 7-substituted tetracycline compound may be reactedwith N-(5-chloro-2-pyridyl)bis(trifluoromethanesulfonimide) to form atrifluoromethane substituted intermediate (14), which can then bereacted with ammonium formate in the presence of a palladium catalyst toform the desired product (15). Compounds D, E, G, H, S and W may besynthesized as shown in Scheme 5.

Scheme 6 outlines the general synthesis of 7-substituted tetracyclines.A 7-iodo sancycline derivative (1) may undergo a Stille coupling or aSuzuki coupling by reacting with an alkyl tin derivative or a boronicacid derivative in the presence of a palladium catalyst to form thedesired product (16). Compounds J, K, L and T may be synthesized asillustrated in Scheme 6.

The 7-substituted oxime derivatives may also be prepared as shown inScheme 7. An 7-iodo sancycline derivative (1) can be reacted with asubstituted alkyne in the presence of palladium to synthesize thealkynyl derivative 17. Compound 17 may be converted to the acylsubstituted compound 18 by any technique known in the art. The desiredoxime product 19 can be obtained by reacting the acyl moiety with aprimary hydroxylamine. Compounds I, O, and AN may be synthesized asshown in Scheme 7.

Scheme 8 is a general synthetic scheme showing the synthesis of7-substituted hydrazone compounds. A 7-substituted aldehyde tetracyclinederivative, prepared as described above in Scheme 4, is combined with aprimary hydrazone to form the desired product 20. Compounds X and AC maybe synthesized as shown in Scheme 8.

7-substituted hydrazines may also be synthesized as shown in Scheme 9.Starting with compound 2, synthesized as described in Scheme 4 above,may be reacted with a secondary hydrazine in the presence of a reducingagent to form compound 21. Compound Z may be synthesized as shown inScheme 9.

Scheme 10 further depicts a method of synthesizing a 7-substitutedaminoalkyl tetracycline compound. Compound 2 is reacted with a primaryamine in the presence of a reducing agent to form the secondary amineintermediate (22), which is then mixed with an acid chloride to formcompound 23. Compounds F, H, CV and CW may be synthesized as illustratedin Scheme 10.

Scheme 11 describes a general method for preparing 9-substitutedaminoalkyl substituted tetracycline compounds. Compound 24 may bereacted directly with a secondary amine to form compounds similar to 26.Alternatively, compound 24 may be mixed with a primary amine to yieldthe substituted imine 25, which may be further reduced to produce theaminoalkyl compound 26. Compounds V, AK and AH may be synthesized asshown in Scheme 11.

7-substituted tetracycline may also be prepared as shown in Scheme 12.Starting again with compound 2, reductive alkylation with a dioxalanylsecondary amine yields the intermediate 27. Subsequently exposing 27 toacidic conditions removes the protecting group to form intermediate 28,which may then be reacted with a primary amine to form product 29.Compound AL may be synthesized as shown in Scheme 12.

Schemes 13 and 14 illustrate the general synthesis of cyclobutene7-substituted tetracycline compounds. Beginning with 30, tin reagent 31is synthesized by reacting 30 with a trimethylsilyl substituted alkyltinderivative.

Scheme 14 continues to show the synthesis of cyclobutenedione7-substituted tetracycline compounds, by reacting building block 31 with7-iodo substituted sancycline (1) in a Stille coupling reaction to form32. The amino substitution of product 33 is accomplished by reacting 32with a primary amine in methanol. Compounds AD, AI and AJ may besynthesized as shown in Schemes 13 and 14.

Scheme 15 depicts generally the synthesis of substituted aromatic7-substituted tetracycline compounds. Beginning with 1 and performing aSuzuki coupling reaction in the presence of a boronic acid and apalladium catalyst, compounds of general formula 34 are formed.Compounds AO, AP, AQ, AR, AS, AT, AU, AV, AW, AX, AY, AZ, BA, BB, BN,DK, DL, DM, DN, DO and DP may be synthesized as shown in Scheme 15.

Scheme 16 also depicts the synthesis of substituted aromatic7-substituted tetracycline compounds. Again, starting from 7-iodosubstituted sancycline (1), a Suzuki coupling reaction is performed witha boronic acid in the presence of a palladium catalyst to provideintermediate 35 in which R^(7tb) is either an amine or a carboxylicacid. If R^(7tb) is a carboxylic acidic moiety, a coupling to asecondary amine in the presence of base and a typical coupling reagentto form 7-substituted tetracyclines similar to 36a. Compounds BC, BD,BE, BF, BG, BH, BI, BJ, BK and DQ may be synthesized as illustrated inthis manner. If R^(7tb) is an amino moiety, a coupling to an acidchloride or carboxylic acid in the presence of a base and a typicalcoupling reagent to form 7-substituted tetracyclines similar to 36b.Compounds BO and BP may be synthesized in this manner.

Scheme 17 depicts a method for synthesizing aromatic substituted9-substituted tetracycline compounds. A 9-iodo tetracycline derivativeis reacted under Suzuki conditions by mixing with a boronic acid in thepresence of the appropriate palladium catalyst to give compounds similarto compound 38. Compounds BL and BM may be synthesized as illustrated asin Scheme 17.

Scheme 18 also depicts a method for synthesizing 9-substitutedtetracycline compounds by reacting 37 with a palladium catalyst andcarbon monoxide in the presence of N-hydroxysuccinimide to generate anactivated ester intermediate. Reaction of this intermediate withnucleophilic compounds such as alcohols, hydroxylamines or amines yieldsthe desired ester, hydroxamic acid or amide, respectively, similar to39. Compounds BQ, BR, BS, BT, BU, BV, BW, BX, BY and BZ were alsosynthesized in a similar manner.

9-substituted oxime tetracycline compounds may be prepared as shown inScheme 19. A 9-iodo substituted tetracycline derivative is subjected toHeck conditions to form an alkyne intermediate which is converted to 40by acid hydrolysis. Intermediate 40 is subsequently reacted with anappropriate hydroxylamine to yield the desired product 41. Compounds CGand CH may be synthesized as shown in Scheme 19.

As shown in Scheme 20, 7-substituted tetracycline compounds can also beprepared by reacting the acyl intermediate 41 with hydrogen bromide toform the α-bromoketone substituted tetracycline 42. By reacting thebrominated tetracycline with a secondary amine, followed by exposure toan acid chloride, the desired product 43 can be formed. Compounds CZ, DAand DB may be synthesized as illustrated in Scheme 20.

As shown in Scheme 21, 9-substituted-4-dedimethylamino tetracyclinecompounds may be synthesized starting from minocycline, which is exposedto the dedimethylamino conditions of methyl iodide and zinc to form4-dedimethylsancycline 44. Intermediate 44 is halogenated at the9-position to form intermediate 45, and upon exposing 45 to theappropriate palladium conditions, compounds similar to 46 are formed.Compounds BQ, BR, BW, BX, BY, BZ, CA, CB, CC, CD, CE, CF, EI, EJ, EK,EL, EM, CI, CJ, CK, CL, EN, EO, EP, EQ, ER, ES, ET, EU, EV, EW, EX, FMand FN may be synthesized as shown in Scheme 21.

As shown in Scheme 22, 9-substituted-4-dedimethylamino doxycyclinecompounds may be synthesized starting from doxycycline, which is exposedto the dedimethylamino conditions of methyl iodide and zinc to form4-dedimethyldoxycycline 47. Intermediate 47 is halogenated at the9-position to form intermediate 48, and upon exposing 48 to theappropriate palladium conditions, compounds similar to 49 are formed.Compound CP may be synthesized as shown in Scheme 22.

Scheme 23 illustrates the synthesis of 10-substituted tetracyclinecompounds. Starting with minocycline, the 10-position hydroxide isdeprotonated in the presence of a strong base, followed by the additionof triflate to form intermediate 50, which then undergoes either Stilleor Suzuki conditions or carbonylation conditions to form compoundssimilar to 10-substituted compounds 51. Compounds CM, CN, CO, EY, EZ,FA, FO, FP and FQ may be synthesized as illustrated in Scheme 23.

Scheme 24 illustrates the synthesis of 7-aminomethyl substitutedtetracycline compounds. Compound 1 is exposed to a secondary amine inthe presence of a reducing agent to from compounds similar to 52.Compounds DC, DD, DE, DF, DG, DH, DI, DJ and FC may be synthesized asillustrated in Scheme 24.

Scheme 25 provides a method for synthesizing 9-aminomethyl substitutedtetracycline compounds. 9-formyl substituted compound 54 is reacted witha secondary amine in the presence of a reducing agent to provide the9-aminomethyl substituted tetracycline compounds 55. Compounds FD, FE,FF, FG, FH, FI, FJ, FK and FL may be synthesized as illustrated inScheme 25.

Scheme 26 illustrates the methods for synthesizing 9-alkyl or 9-carbonylsubstituted tetracycline compounds starting with the 9-iodominocyclineor 9-iodo-4-dedimethylminocycline compound 56, followed by palladiumcatalyzed alkynylation to form intermediate 57. Intermediate 57 mayundergo either hydrogenolysis to form 9-alkyl substituted tetracyclinecompounds (58) or acid catalyzed hydrolysis to form 9-carbonylsubstituted tetracycline compound (59). Compounds FR, FS, FT and FU maybe prepared as illustrated in Scheme 26.

9-Alkyl substituted tetracycline compounds may also be synthesized asshown in Scheme 27. Again starting with 9-iodominocycline or9-iodo-4-dedimethylminocycline compound 56, either Suzuki or Stillecoupling conditions may produce the 9-alkyl substituted tetracyclinecompounds (60) or reaction with copper iodide with a fluorinated estercompound yields 9-trifluoroalkyl substituted compound 61. Compounds FV,FW, FX, FY, GA and GB may be synthesized as illustrated in Scheme 27.

7-Furanyl substituted tetracycline compounds may be synthesized asillustrated in Scheme 28. 7-Iodosancycline (1) is subjected to a formylsubstituted furanyl boronic acid in the presence of palladium (II)acetate and sodium carbonate to yield intermediate 62. A reductiveamination is then performed in the presence of an appropriate reducingagent and a secondary amine to convert the formyl moiety to a tertiaryalkylamine (63). The substituents of the tertiary amine may be furtherderivitized, as shown by the reaction of compound 63 withmethylchloroformate to form compound 64. Compounds FB, CQ, CR, CS and CTmay be synthesized as shown in Scheme 28.

7-Pyridinyl-9-aminocarbonyl substituted tetracyclines may be synthesizedas shown in Scheme 29. The 7-position of the 7-iodo-9-nitro tetracyclinecompound (8) is reacted under Stille conditions to form the 7-pyridinylintermediate 64, which is then subjected to reducing conditions to formthe 7-pyridinyl-9-amino tetracycline compound 65. The amino moiety ofcompound 65 is then reacted with a chloroformate to form the desiredaminocarbonyl substituent in the 9 position (66). Compounds GS, GT, GUand GV may also be synthesized as shown in Scheme 29.

Scheme 30 illustrates methods for preparing both 9-aminomethylsubstituted sancycline compounds and 7-substituted-9-aminomethyltetracycline compounds. Sancycline is bromonated at the 7-position withN-bromosuccinimide and iodated at the 9-position with N-iodosuccinimideto form the dihalogenated reactive intermediate 67, which then undergoesformylation at the 9-position to yield a 7-bromo-9-formyl substitutedtetracycline compound (68). Compound 68 may then undergo a reductiveamination procedure in the presence of an appropriate secondary amineand a reducing agent to form compound 69. The bromo moiety at the7-position may then be removed by exposing 69 to palladium on carbon inthe presence of hydrogen gas to provide 9-aminomethyl sancyclinecompounds (71). Alternatively, the reactive intermediate 68 may first beexposed to reductive amination conditions as described above, followedby a pallium-indium cross coupling reaction to form7-substituted-9-aminomethyl tetracycline compounds (70). Compounds GN,GO, GP and GQ may be synthesized as shown in Scheme 30.

7-Substituted tetracycline compounds may be generally synthesized asshown in Scheme 31. A 7-iodo tetracycline compound (71) may be reactedunder Suzuki, Stille or indium-palladium cross coupling reactions toform 7-substituted tetracycline compounds. Compounds GC, GD, GE, GF andGH may be synthesized as shown in Scheme 31.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.The term alkyl further includes alkyl groups, which can further includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkyl has 6 or fewer carbon atoms in itsbackbone (e.g., C₁-C₆ for straight chain, C₃-C₆ for branched chain), andmore preferably 4 or fewer. Likewise, preferred cycloalkyls have from3-8 carbon atoms in their ring structure, and more preferably have 5 or6 carbons in the ring structure. The term C₁-C₆ includes alkyl groupscontaining 1 to 6 carbon atoms.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls,” the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “arylalkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). The term “alkyl” also includes the side chains of natural andunnatural amino acids.

The term “aryl” includes groups, including 5- and 6-membered single-ringaromatic groups that may include from zero to four heteroatoms, forexample, benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles,” “heterocycles,” “heteroaryls” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, arylalkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings which are not aromatic so as to form apolycycle (e.g., tetralin).

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond.

For example, the term “alkenyl” includes straight-chain alkenyl groups(e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl,octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups,cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substitutedcycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenylgroups. The term alkenyl further includes alkenyl groups which includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkenyl group has 6 or fewer carbon atoms in itsbackbone (e.g., C₂-C₆ or straight chain, C₃-C₆ for branched chain).Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in theirring structure, and more preferably have 5 or 6 carbons in the ringstructure. The term C₂-C₆ includes alkenyl groups containing 2 to 6carbon atoms.

Moreover, the term alkenyl includes both “unsubstituted alkenyls” and“substituted alkenyls,” the latter of which refers to alkenyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond.

For example, the term “alkynyl” includes straight-chain alkynyl groups(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl,nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkylor cycloalkenyl substituted alkynyl groups. The term alkynyl furtherincludes alkynyl groups which include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of the hydrocarbonbackbone. In certain embodiments, a straight chain or branched chainalkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C₂-C₆for straight chain, C₃-C₆ for branched chain). The term C₂-C₆ includesalkynyl groups containing 2 to 6 carbon atoms.

Moreover, the term alkynyl includes both “unsubstituted alkynyls” and“substituted alkynyls,” the latter of which refers to alkynyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxyearbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto five carbon atoms in its backbone structure. “Lower alkenyl” and“lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.

The term “acyl” includes compounds and moieties which contain the acylradical (CH₃CO—) or a carbonyl group. It includes substituted acylmoieties. The term “substituted acyl” includes acyl groups where one ormore of the hydrogen atoms are replaced by for example, alkyl groups,alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxyearbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “acylamino” includes moieties wherein an acyl moiety is bondedto an amino group. For example, the term includes alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido groups.

The term “aroyl” includes compounds and moieties with an aryl orheteroaromatic moiety bound to a carbonyl group. Examples of aroylgroups include phenylcarboxy, naphthyl carboxy, etc.

The terms “alkoxyalkyl,” “alkylaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withgroups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties. Examples ofhalogen substituted alkoxy groups include, but are not limited to,fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,dichloromethoxy, trichloromethoxy, etc.

The term “amine” or “amino” includes compounds where a nitrogen atom iscovalently bonded to at least one carbon or heteroatom. The termincludes “alkyl amino” which comprises groups and compounds wherein thenitrogen is bound to at least one additional alkyl group. The term“dialkyl amino” includes groups wherein the nitrogen atom is bound to atleast two additional alkyl groups. The term “arylamino” and“diarylamino” include groups wherein the nitrogen is bound to at leastone or two aryl groups, respectively. The term “alkylarylamino,”“alkylaminoaryl” or “arylaminoalkyl” refers to an amino group which isbound to at least one alkyl group and at least one aryl group. The term“alkaminoalkyl” refers to an alkyl, alkenyl, or alkynyl group bound to anitrogen atom which is also bound to an alkyl group.

The term “amide,” “amido” or “aminocarbonyl” includes compounds ormoieties which contain a nitrogen atom which is bound to the carbon of acarbonyl or a thiocarbonyl group. The term includes “alkaminocarbonyl”or “alkylaminocarbonyl” groups which include alkyl, alkenyl, aryl oralkynyl groups bound to an amino group bound to a carbonyl group. Itincludes arylaminocarbonyl and arylcarbonylamino groups which includearyl or heteroaryl moieties bound to an amino group which is bound tothe carbon of a carbonyl or thiocarbonyl group. The terms“alkylaminocarbonyl,” “alkenylaminocarbonyl,” “alkynylaminocarbonyl,”“arylaminocarbonyl,” “alkylcarbonylamino,” “alkenylcarbonylamino,”“alkynylcarbonylamino,” and “arylcarbonylamino” are included in term“amide.” Amides also include urea groups (aminocarbonylamino) andcarbamates (oxycarbonylamino).

The term “carbonyl” or “carboxy” includes compounds and moieties whichcontain a carbon connected with a double bond to an oxygen atom. Thecarbonyl can be further substituted with any moiety which allows thecompounds of the invention to perform its intended function. Forexample, carbonyl moieties may be substituted with alkyls, alkenyls,alkynyls, aryls, alkoxy, aminos, etc. Examples of moieties which containa carbonyl include aldehydes, ketones, carboxylic acids, amides, esters,anhydrides, etc.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The term “ester” includes compounds and moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc. The alkyl, alkenyl, or alkynyl groups are asdefined above.

The term “thioether” includes compounds and moieties which contain asulfur atom bonded to two different carbon or hetero atoms. Examples ofthioethers include, but are not limited to alkthioalkyls,alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” includecompounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfuratom which is bonded to an alkyl group. Similarly, the term“alkthioalkenyls” and alkthioalkynyls” refer to compounds or moietieswherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atomwhich is covalently bonded to an alkynyl group.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

The terms “polycyclyl” or “polycyclic radical” refer to two or morecyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls) in which two or more carbons are common to twoadjoining rings, e.g., the rings are “fused rings.” Rings that arejoined through non-adjacent atoms are termed “bridged” rings. Each ofthe rings of the polycycle can be substituted with such substituents asdescribed above, as for example, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, alkylaminoacaibonyl,arylalkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,arylcarbonyl, arylalkyl carbonyl, alkenylcarbonyl, aminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amido, amino (including alkyl amino, dialkylamino, arylamino,diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or anaromatic or heteroaromatic moiety.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “prodrug moiety” includes moieties which can be metabolized invivo to a hydroxyl group and moieties which may advantageously remainesterified in vivo. Preferably, the prodrugs moieties are metabolized invivo by esterases or by other mechanisms to hydroxyl groups or otheradvantageous groups. Examples of prodrugs and their uses are well knownin the art (See, e.g., Berge et al. (1977) “Pharmaceutical Salts” J.Pharm. Sci. 66:1-19). The prodrugs can be prepared in situ during thefinal isolation and purification of the compounds, or by separatelyreacting the purified compound in its free acid form or hydroxyl with asuitable esterifying agent. Hydroxyl groups can be converted into estersvia treatment with a carboxylic acid. Examples of prodrug moietiesinclude substituted and unsubstituted, branch or unbranched lower alkylester moieties, (e.g., propionoic acid esters), lower alkenyl esters,di-lower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethylester), acylamino lower alkyl esters (e.g., acetyloxymethyl ester),acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters(phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester),substituted (e.g., with methyl, halo, or methoxy substituents) aryl andaryl-lower alkyl esters, amides, lower-alkyl amides, di-lower alkylamides, and hydroxy amides. Preferred prodrug moieties are propionoicacid esters and acyl esters.

It will be noted that the structure of some of the tetracyclinecompounds of this invention includes asymmetric carbon atoms. It is tobe understood accordingly that the isomers arising from such asymmetry(e.g., all enantiomers and diastereomers) are included within the scopeof this invention, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis. Furthermore, thestructures and other compounds and moieties discussed in thisapplication also include all tautomers thereof.

Methods for Treating Tetracycline Responsive States

The invention also pertains to methods for treating a tetracyclineresponsive states in subjects, by administering to a subject aneffective amount of a tetracycline compound of the invention (e.g., acompound of Formula I, II, III, Ilia, IV, IVa, V, Va, VI, VII, VIIa,VIII, VIIIa, IX, IXa, X, Xa, XT, XIa, XII, XIII, XIV, XV or XVI, or acompound listed in Table 2 or otherwise described herein), such that thetetracycline responsive state is treated.

The term “treating” includes curing as well as ameliorating at least onesymptom of the state, disease or disorder, e.g., the tetracyclinecompound responsive state.

The language “tetracycline compound responsive state” or “tetracyclineresponsive state” includes states which can be treated, prevented, orotherwise ameliorated by the administration of a tetracycline compoundof the invention Tetracycline compound responsive states includebacterial, viral, parasitic, and fungal infections (including thosewhich are resistant to other tetracycline compounds), cancer (e.g.,prostate, breast, colon, lung melanoma and lymph cancers and otherdisorders characterized by unwanted cellular proliferation, including,but not limited to, those described in U.S. Pat. No. 6,100,248),arthritis, osteoporosis, diabetes, stroke, AMI, aortic aneurysm,neurodegenerative diseases and other states for which tetracyclinecompounds have been found to be active (see, for example, U.S. Pat. Nos.5,789,395; 5,834,450; 6,277,061 and 5,532,227, each of which isexpressly incorporated herein by reference). Compounds of the inventioncan be used to prevent or control important mammalian and veterinarydiseases such as diarrhea, urinary tract infections, infections of skinand skin structure, ear, nose and throat infections, wound infection,mastitis and the like. In addition, methods for treating neoplasms usingtetracycline compounds of the invention are also included (van derBozert et al., Cancer Res., 48:6686-6690 (1988)). In a furtherembodiment, the tetracycline responsive state is not a bacterialinfection. In another embodiment, the tetracycline compounds of theinvention are essentially non-antibacterial. For example,non-antibacterial tetracycline compounds of the invention may have MICvalues greater than about 4 μg/ml (as measured by assays known in theart and/or the assay given in Example 2).

Tetracycline compound responsive states also include inflammatoryprocess associated states (IPAS). The term “inflammatory processassociated state” includes states in which inflammation or inflammatoryfactors (e.g., matrix metalloproteinases (MMPs), nitric oxide (NO), TNF,interleukins, plasma proteins, cellular defense systems, cytokines,lipid metabolites, proteases, toxic radicals, adhesion molecules, etc.),are involved or are present in an area in aberrant amounts, e.g., inamounts which may be advantageous to alter, e.g., to benefit thesubject. The term “inflammatory process associated state” also includesstates in which there is an increase in acute phase proteins (e.g.,C-reactive protein). The inflammatory process is the response of livingtissue to damage. The cause of inflammation may be due to physicaldamage, chemical substances, micro-organisms, tissue necrosis, cancer orother agents. Acute inflammation is short-lasting, lasting only a fewdays. If it is longer lasting however, then it may be referred to aschronic inflammation.

Tetracycline responsive states also include inflammatory disorders.Inflammatory disorders are generally characterized by heat, redness,swelling, pain and loss of function. Examples of causes of inflammatorydisorders include, but are not limited to, microbial infections (e.g.,bacterial and fungal infections), physical agents (e.g., burns,radiation, and trauma), chemical agents (e.g., toxins and causticsubstances), tissue necrosis and various types of immunologic reactions.

Examples of inflammatory disorders include, but are not limited to,osteoarthritis, rheumatoid arthritis, acute and chronic infections(bacterial and fungal, including diphtheria and pertussis); acute andchronic bronchitis, sinusitis, and upper respiratory infections,including the common cold; acute and chronic gastroenteritis andcolitis; acute and chronic cystitis and urethritis; acute and chronicdermatitis; acute and chronic conjunctivitis; acute and chronicserositis (pericarditis, peritonitis, synovitis, pleuritis andtendinitis); uremic pericarditis; acute and chronic cholecystis; acuteand chronic vaginitis; acute and chronic uveitis; drug reactions; animalbites (e.g., spider bites, snake bites, insect bites and the like);burns (thermal, chemical, and electrical); inflammatory bowel disorder(IBD); common obstructive pulmonary disease (COPD); acute respiratorydistress syndrome (ARDS); vasculitis; asthma; sepsis; nephritis;pancreatitis; hepatitis; lupus; viral infections; parasitic infections;and sunburn.

Tetracycline compound responsive states also include NO associatedstates. The term “NO associated state” includes states which involve orare associated with nitric oxide (NO) or inducible nitric oxide synthase(iNOS). NO associated state includes states which are characterized byaberrant amounts of NO and/or iNOS. Preferably, the NO associated statecan be treated by administering tetracycline compounds of the invention.The disorders, diseases and states described in U.S. Pat. Nos.6,231,894; 6,015,804; 5,919,774; and 5,789,395 are also included as NOassociated states. The entire contents of each of these patents arehereby incorporated herein by reference.

Other examples of tetracycline compound responsive states include, butare not limited to, malaria, senescence, diabetes, vascular stroke,hemorrhagic stroke, neurodegenerative disorders (Alzheimer's disease &Huntington's disease), cardiac disease (reperfusion-associated injuryfollowing infarction), juvenile diabetes, inflammatory disorders,osteoarthritis, rheumatoid arthritis, acute, recurrent and chronicinfections (bacterial, viral and fungal); acute and chronic bronchitis,sinusitis, and respiratory infections, including the common cold; acuteand chronic gastroenteritis and colitis; acute and chronic cystitis andurethritis; acute and chronic dermatitis; acute and chronicconjunctivitis; acute and chronic serositis (pericarditis, peritonitis,synovitis, pleuritis and tendonitis); uremic pericarditis; acute andchronic cholecystis; cystic fibrosis, acute and chronic vaginitis; acuteand chronic uveitis; drug reactions; insect bites; burns (thermal,chemical, and electrical); and sunburn.

The term “inflammatory process associated state” also includes, in oneembodiment, matrix metalloproteinase associated states (MMPAS). MMPASinclude states characterized by aberrant amounts of MMPs or MMPactivity. These are also include as tetracycline compound responsivestates which may be treated using compounds of the invention.

Examples of other tetracycline compound responsive states include, butare not limited to, arteriosclerosis, angiogenesis, corneal ulceration,emphysema, osteoarthritis, multiple sclerosis (Liedtke et al., Ann.Neurol. 1998, 44:35-46; Chandler et al., J. Neuroimmunol. 1997,72:155-71), osteosarcoma, osteomyelitis, bronchiectasis, chronicpulmonary obstructive disease, skin and eye diseases, periodontitis,osteoporosis, rheumatoid arthritis, ulcerative colitis, inflammatorydisorders, tumor growth and invasion (Stetler-Stevenson et al., Annu.Rev. Cell Biol. 1993, 9:541-73; Tryggvason et al., Biochim. Biophys.Acta 1987, 907:191-217; Li et al., Mol. Carcinog. 1998, 22:84-89)),metastasis, acute lung injury, stroke, ischemia, diabetes, aortic orvascular aneurysms, skin tissue wounds, dry eye, bone and cartilagedegradation (Greenwald el al., Bone 1998, 22:33-38; Ryan et al., Curr.Op. Rheumatol. 1996, 8; 238-247). Other MMPAS include those described inU.S. Pat. Nos. 5,459,135; 5,321,017; 5,308,839; 5,258,371; 4,935,412;4,704,383, 4,666,897, and RE 34,656, incorporated herein by reference intheir entirety.

In another embodiment, the tetracycline compound responsive state iscancer. Examples of cancers which the tetracycline compounds of theinvention may be useful to treat include all solid tumors, i.e.,carcinomas e.g., adenocarcinomas, and sarcomas. Adenocarcinomas arecarcinomas derived from glandular tissue or in which the tumor cellsform recognizable glandular structures. Sarcomas broadly include tumorswhose cells are embedded in a fibrillar or homogeneous substance likeembryonic connective tissue. Examples of carcinomas which may be treatedusing the methods of the invention include, but are not limited to,carcinomas of the prostate, breast, ovary, testis, lung, colon, andbreast. The methods of the invention are not limited to the treatment ofthese tumor types, but extend to any solid tumor derived from any organsystem. Examples of treatable cancers include, but are not limited to,colon cancer, bladder cancer, breast cancer, melanoma, ovariancarcinoma, prostatic carcinoma, lung cancer, and a variety of othercancers as well. The methods of the invention also cause the inhibitionof cancer growth in adenocarcinomas, such as, for example, those of theprostate, breast, kidney, ovary, testes, and colon.

In an embodiment, the tetracycline responsive state of the invention iscancer. The invention pertains to a method for treating a subjectsuffering or at risk of suffering from cancer, by administering aneffective amount of a substituted tetracycline compound, such thatinhibition cancer cell growth occurs, i.e., cellular proliferation,invasiveness, metastasis, or tumor incidence is decreased, slowed, orstopped. The inhibition may result from inhibition of an inflammatoryprocess, down-regulation of an inflammatory process, some othermechanism, or a combination of mechanisms. Alternatively, thetetracycline compounds may be useful for preventing cancer recurrence,for example, to treat residual cancer following surgical resection orradiation therapy. The tetracycline compounds useful according to theinvention are especially advantageous as they are substantiallynon-toxic compared to other cancer treatments. In a further embodiment,the compounds of the invention are administered in combination withstandard cancer therapy, such as, but not limited to, chemotherapy.

Other examples of tetracycline compound responsive states includeneurological disorders which include both neuropsychiatric andneurodegenerative disorders, but are not limited to, such as Alzheimer'sdisease, dementias related to Alzheimer's disease (such as Pick'sdisease), Parkinson's and other Lewy diffuse body diseases, seniledementia, Huntington's disease, Gilles de la Tourette's syndrome,multiple sclerosis (e.g., including but not limited to, relapsing andremitting multiple sclerosis, primary progressive multiple sclerosis,and secondary progressive multiple sclerosis), amyotrophic lateralsclerosis (ALS), progressive supranuclear palsy, epilepsy, andCreutzfeldt-Jakob disease; autonomic function disorders such ashypertension and sleep disorders, and neuropsychiatric disorders, suchas depression, schizophrenia, schizoaffective disorder, Korsakoff spsychosis, mania, anxiety disorders, or phobic disorders; learning ormemory disorders, e.g., amnesia or age-related memory loss, attentiondeficit disorder, dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), bipolar affectiveneurological disorders, e.g., migraine and obesity. Further neurologicaldisorders include, for example, those listed in the American PsychiatricAssociation's Diagnostic and Statistical manual of Mental Disorders(DSM), the most current version of which is incorporated herein byreference in its entirety. Other examples of tetracycline compoundresponsive states are described in WO 03/005971A2, U.S. Ser. No.60/421,248, and U.S. Ser. No. 60/480,482, each incorporated herein byreference.

The language “in combination with” another therapeutic agent ortreatment includes co-administration of the tetracycline compound,(e.g., inhibitor) and with the other therapeutic agent or treatment,administration of the tetracycline compound first, followed by the othertherapeutic agent or treatment and administration of the othertherapeutic agent or treatment first, followed by the tetracyclinecompound. The other therapeutic agent may be any agent that is known inthe art to treat, prevent, or reduce the symptoms of a particulartetracycline responsive state. Furthermore, the other therapeutic agentmay be any agent of benefit to the patient when administered incombination with the administration of a tetracycline compound. In oneembodiment, the cancers treated by methods of the invention includethose described in U.S. Pat. Nos. 6,100,248; 5,843,925; 5,837,696; or5,668,122, incorporated herein by reference in their entirety.

In another embodiment, the tetracycline compound responsive state isdiabetes, e.g., juvenile diabetes, diabetes mellitus, diabetes type I,or diabetes type II. In a further embodiment, protein glycosylation isnot affected by the administration of the tetracycline compounds of theinvention. In another embodiment, the tetracycline compound of theinvention is administered in combination with standard diabetictherapies, such as, but not limited to insulin therapy. In a furtherembodiment, the IPAS includes disorders described in U.S. Pat. Nos.5,929,055; and 5,532,227, incorporated herein by reference in theirentirety.

In another embodiment, the tetracycline compound responsive state is abone mass disorder. Bone mass disorders include disorders are disordersand states where the formation, repair or remodeling of bone isadvantageous. For examples bone mass disorders include osteoporosis(e.g., a decrease in bone strength and density), bone fractures, boneformation associated with surgical procedures (e.g., facialreconstruction), osteogenesis imperfecta (brittle bone disease),hypophosphatasia, Paget's disease, fibrous dysplasia, osteopetrosis,myeloma bone disease, and the depletion of calcium in bone, such as thatwhich is related to primary hyperparathyroidism. Bone mass disordersinclude all states in which the formation, repair or remodeling of boneis advantageous to the subject as well as all other disorders associatedwith the bones or skeletal system of a subject which can be treated withthe tetracycline compounds of the invention. In a further embodiment,the bone mass disorders include those described in U.S. Pat. Nos.5,459,135; 5,231,017; 5,998,390; 5,770,588; RE 34,656; 5,308,839;4,925,833; 3,304,227; and 4,666,897, each of which is herebyincorporated herein by reference in its entirety.

In another embodiment, the tetracycline compound responsive state isacute lung injury. Acute lung injuries include adult respiratorydistress syndrome (ARDS), post-pump syndrome (PPS), adelectasis (e.g.,collapsed lung) and trauma. Trauma includes any injury to living tissuecaused by an extrinsic agent or event. Examples of trauma include, butare not limited to, crush injuries, contact with a hard surface, orcutting or other damage to the lungs.

The invention also pertains to a method for treating acute lung injuryby administering a substituted tetracycline compound of the invention.

The tetracycline responsive states of the invention also include chroniclung disorders. The invention pertains to methods for treating chroniclung disorders by administering a tetracycline compound, such as thosedescribed herein. The method includes administering to a subject aneffective amount of a substituted tetracycline compound such that thechronic lung disorder is treated. Examples of chronic lung disordersinclude, but are not limited, to asthma, chronic obstructive pulmonarydisease (COPD), cystic fibrosis, and emphesema. In a further embodiment,the tetracycline compounds of the invention used to treat acute and/orchronic lung disorders such as those described in U.S. Pat. Nos.5,977,091; 6,043,231; 5,523,297; and 5,773,430, each of which is herebyincorporated herein by reference in its entirety.

In yet another embodiment, the tetracycline compound responsive state isischemia, stroke, hemorrhagic stroke or ischemic stroke. The inventionalso pertains to a method for treating ischemia, stroke, hemorrhagicstroke or ischemic stroke by administering an effective amount of asubstituted tetracycline compound of the invention. In a furtherembodiment, the tetracycline compounds of the invention are used totreat such disorders as described in U.S. Pat. Nos. 6,231,894;5,773,430; 5,919,775 or 5,789,395, incorporated herein by reference.

In another embodiment, the tetracycline compound responsive state is askin wound. The invention also pertains, at least in part, to a methodfor improving the healing response of the epithelialized tissue (e.g.,skin, mucusae) to acute traumatic injury (e.g., cut, burn, scrape,etc.). The method may include using a tetracycline compound of theinvention (which may or may not have antibacterial activity) to improvethe capacity of the epithelialized tissue to heal acute wounds. Themethod may increase the rate of collagen accumulation of the healingtissue. The method may also decrease the proteolytic activity in theepthithelialized tissue by decreasing the collagenolytic and/orgellatinolytic activity of MMPs. In a further embodiment, thetetracycline compound of the invention is administered to the surface ofthe skin (e.g., topically). In a further embodiment, the tetracyclinecompound of the invention used to treat a skin wound, and other suchdisorders as described in, for example, U.S. Pat. Nos. 5,827,840;4,704,383; 4,935,412; 5,258,371; 5,308,8391 5,459,135; 5,532,227; and6,015,804; each of which is incorporated herein by reference in itsentirety.

In yet another embodiment, the tetracycline compound responsive state isan aortic or vascular aneurysm in vascular tissue of a subject (e.g., asubject having or at risk of having an aortic or vascular aneurysm,etc.). The tetracycline compound may by effective to reduce the size ofthe vascular aneurysm or it may be administered to the subject prior tothe onset of the vascular aneurysm such that the aneurysm is prevented.In one embodiment, the vascular tissue is an artery, e.g., the aorta,e.g., the abdominal aorta. In a further embodiment, the tetracyclinecompounds of the invention are used to treat disorders described in U.S.Pat. Nos. 6,043,225 and 5,834,449, incorporated herein by reference intheir entirety.

Bacterial infections may be caused by a wide variety of gram positiveand gram negative bacteria. Some of the compounds of the invention areuseful as antibiotics against organisms which are resistant and/orsensitive to other tetracycline compounds. The antibiotic activity ofthe tetracycline compounds of the invention may by using the in vitrostandard broth dilution method described in Waitz, J. A., CISI, DocumentM7-A2, vol. 10, no. 8, pp. 13-20, 2^(nd) edition, Villanova, Pa. (1990).

The tetracycline compounds may also be used to treat infectionstraditionally treated with tetracycline compounds such as, for example,rickettsiae; a number of gram-positive and gram-negative bacteria; andthe agents responsible for lymphogranuloma venereum, inclusionconjunctivitis, or psittacosis. The tetracycline compounds may be usedto treat infections of, e.g., K. pneumoniae. Salmonella, E. hirae, A.baumanii, B. catarrhalis, H. influenzae. P. aeruginosa, E. faecium, E.coli. S. aureus or E. faecalis. In one embodiment, the tetracyclinecompound is used to treat a bacterial infection that is resistant toother tetracycline antibiotic compounds. The tetracycline compound ofthe invention may be administered with a pharmaceutically acceptablecarrier.

The language “effective amount” of the compound is that amount necessaryor sufficient to treat or prevent a tetracycline compound responsivestate. The effective amount can vary depending on such factors as thesize and weight of the subject, the type of illness, or the particulartetracycline compound. For example, the choice of the tetracyclinecompound can affect what constitutes an “effective amount.” One ofordinary skill in the art would be able to study the aforementionedfactors and make the determination regarding the effective amount of thetetracycline compound without undue experimentation.

The invention also pertains to methods of treatment againstmicroorganism infections and associated diseases. The methods includeadministration of an effective amount of one or more tetracyclinecompounds to a subject. The subject can be either a plant or,advantageously, an animal, e.g., a mammal, e.g., a human.

In the therapeutic methods of the invention, one or more tetracyclinecompounds of the invention may be administered alone to a subject, ormore typically a compound of the invention will be administered as partof a pharmaceutical composition in mixture with conventional excipient,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for parenteral, oral or other desired administrationand which do not deleteriously react with the active compounds and arenot deleterious to the recipient thereof.

Pharmaceutical Compositions of the Invention

The invention also pertains to pharmaceutical compositions comprising atherapeutically effective amount of a tetracycline compound (e.g., acompound of Formula I, II, III, IIIa, IV, IVa, V, Va, VI, VII, VIIa,VIII, VIIIa, IX, IXa, X, Xa, XI, Xa, XII, XIII, XIV, XV or XVI or acompound listed in Table 2 or any other compound described herein) and,optionally, a pharmaceutically acceptable carrier.

The language “pharmaceutically acceptable carrier” includes substancescapable of being coadministered with the tetracycline compound(s), andwhich allow both to perform their intended function, e.g., treat orprevent a tetracycline responsive state. Suitable pharmaceuticallyacceptable carriers include but are not limited to water, saltsolutions, alcohol, vegetable oils, polyethylene glycols, gelatin,lactose, amylose, magnesium stearate, talc, silicic acid, viscousparaffin, perfume oil, fatty acid monoglycerides and diglycerides,petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds of the invention.

The tetracycline compounds of the invention that are basic in nature arecapable of forming a wide variety of salts with various inorganic andorganic acids. The acids that may be used to prepare pharmaceuticallyacceptable acid addition salts of the tetracycline compounds of theinvention that are basic in nature are those that form non-toxic acidaddition salts, i.e., salts containing pharmaceutically acceptableanions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate,sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate,lactate, salicylate, citrate, acid citrate, tartrate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand palmoate [i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)] salts.Although such salts must be pharmaceutically acceptable foradministration to a subject, e.g., a mammal, it is often desirable inpractice to initially isolate a tetracycline compound of the inventionfrom the reaction mixture as a pharmaceutically unacceptable salt andthen simply convert the latter back to the free base compound bytreatment with an alkaline reagent and subsequently convert the latterfree base to a pharmaceutically acceptable acid addition salt. The acidaddition salts of the base compounds of this invention are readilyprepared by treating the base compound with a substantially equivalentamount of the chosen mineral or organic acid in an aqueous solventmedium or in a suitable organic solvent, such as methanol or ethanol.Upon careful evaporation of the solvent, the desired solid salt isreadily obtained. The preparation of other tetracycline compounds of theinvention not specifically described in the foregoing experimentalsection can be accomplished using combinations of the reactionsdescribed above that will be apparent to those skilled in the art.

The preparation of other tetracycline compounds of the invention notspecifically described in the foregoing experimental section can beaccomplished using combinations of the reactions described above thatwill be apparent to those skilled in the art.

The tetracycline compounds of the invention that are acidic in natureare capable of forming a wide variety of base salts. The chemical basesthat may be used as reagents to prepare pharmaceutically acceptable basesalts of those tetracycline compounds of the invention that are acidicin nature are those that form non-toxic base salts with such compounds.Such non-toxic base salts include, but are not limited to those derivedfrom such pharmaceutically acceptable cations such as alkali metalcations (e.g., potassium and sodium) and alkaline earth metal cations(e.g., calcium and magnesium), ammonium or water-soluble amine additionsalts such as N-methylglucamine-(meglumine), and the loweralkanolammonium and other base salts of pharmaceutically acceptableorganic amines. The pharmaceutically acceptable base addition salts oftetracycline compounds of the invention that are acidic in nature may beformed with pharmaceutically acceptable cations by conventional methods.Thus, these salts may be readily prepared by treating the tetracyclinecompound of the invention with an aqueous solution of the desiredpharmaceutically acceptable cation and evaporating the resultingsolution to dryness, preferably under reduced pressure. Alternatively, alower alkyl alcohol solution of the tetracycline compound of theinvention may be mixed with an alkoxide of the desired metal and thesolution subsequently evaporated to dryness.

The preparation of other tetracycline compounds of the invention notspecifically described in the foregoing experimental section can beaccomplished using combinations of the reactions described above thatwill be apparent to those skilled in the art.

The tetracycline compounds of the invention and pharmaceuticallyacceptable salts thereof can be administered via either the oral,parenteral or topical routes. In general, these compounds are mostdesirably administered in effective dosages, depending upon the weightand condition of the subject being treated and the particular route ofadministration chosen. Variations may occur depending upon the speciesof the subject being treated and its individual response to saidmedicament, as well as on the type of pharmaceutical formulation chosenand the time period and interval at which such administration is carriedout.

The pharmaceutical compositions of the invention may be administeredalone or in combination with other known compositions for treatingtetracycline responsive states in a subject, e.g., a mammal. Preferredmammals include pets (e.g., cats, dogs, ferrets, etc.), farm animals(cows, sheep, pigs, horses, goats, etc.), lab animals (rats, mice,monkeys, etc.), and primates (chimpanzees, humans, gorillas). Thelanguage “in combination with” a known composition is intended toinclude simultaneous administration of the composition of the inventionand the known composition, administration of the composition of theinvention first, followed by the known composition and administration ofthe known composition first, followed by the composition of theinvention.

The tetracycline compounds of the invention may be administered alone orin combination with pharmaceutically acceptable carriers or diluents byany of the routes previously mentioned, and the administration may becarried out in single or multiple doses. For example, the noveltherapeutic agents of this invention can be administered advantageouslyin a wide variety of different dosage forms, i.e., they may be combinedwith various pharmaceutically acceptable inert carriers in the form oftablets, capsules, lozenges, troches, hard candies, powders, sprays(e.g., aerosols, etc.), creams, salves, suppositories, jellies, gels,pastes, lotions, ointments, aqueous suspensions, injectable solutions,elixirs, syrups, and the like. Such carriers include solid diluents orfillers, sterile aqueous media and various non-toxic organic solvents,etc. Moreover, oral pharmaceutical compositions can be suitablysweetened and/or flavored. In general, the therapeutically-effectivecompounds of this invention are present in such dosage forms atconcentration levels ranging from about 5.0% to about 70% by weight.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch (and preferably corn, potato or tapioca starch), alginicacid and certain complex silicates, together with granulation binderslike polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may also be employed as fillers in gelatin capsules;preferred materials in this connection also include lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the active ingredient may be combined with various sweetening orflavoring agents, coloring matter or dyes, and, if so desired,emulsifying and/or suspending agents as well, together with suchdiluents as water, ethanol, propylene glycol, glycerin and various likecombinations thereof. The compositions of the invention may beformulated such that the tetracycline compositions are released over aperiod of time after administration.

For parenteral administration (including intraperitoneal, subcutaneous,intravenous, intradermal or intramuscular injection), solutions of atherapeutic compound of the present invention in either sesame or peanutoil or in aqueous propylene glycol may be employed. The aqueoussolutions should be suitably buffered (preferably pH greater than 8) ifnecessary and the liquid diluent first rendered isotonic. These aqueoussolutions are suitable for intravenous injection purposes. The oilysolutions are suitable for intraarticular, intramuscular andsubcutaneous injection purposes. The preparation of all these solutionsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well known to those skilled in the art. Forparenteral application, examples of suitable preparations includesolutions, preferably oily or aqueous solutions as well as suspensions,emulsions, or implants, including suppositories. Therapeutic compoundsmay be formulated in sterile form in multiple or single dose formatssuch as being dispersed in a fluid carrier such as sterile physiologicalsaline or 5% saline dextrose solutions commonly used with injectables.

Additionally, it is also possible to administer the compounds of thepresent invention topically when treating inflammatory conditions of theskin. Examples of methods of topical administration include transdermal,buccal or sublingual application. For topical applications, therapeuticcompounds can be suitably admixed in a pharmacologically inert topicalcarrier such as a gel, an ointment, a lotion or a cream. Such topicalcarriers include water, glycerol, alcohol, propylene glycol, fattyalcohols, triglycerides, fatty acid esters, or mineral oils. Otherpossible topical carriers are liquid petrolatum, isopropylpalmitate,polyethylene glycol, ethanol 95%, polyoxyethylene monolauriate 5% inwater, sodium lauryl sulfate 5% in water, and the like. In addition,materials such as anti-oxidants, humectants, viscosity stabilizers andthe like also may be added if desired.

For enteral application, particularly suitable are tablets, dragees orcapsules having talc and/or carbohydrate carrier binder or the like, thecarrier preferably being lactose and/or corn starch and/or potatostarch. A syrup, elixir or the like can be used wherein a sweetenedvehicle is employed. Sustained release compositions can be formulatedincluding those wherein the active component is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc.

In addition to treatment of human subjects, the therapeutic methods ofthe invention also will have significant veterinary applications, e.g.,for treatment of livestock such as cattle, sheep, goats, cows, swine andthe like; poultry such as chickens, ducks, geese, turkeys and the like;horses; and pets such as dogs and cats. Also, the compounds of theinvention may be used to treat non-animal subjects, such as plants.

It will be appreciated that the actual preferred amounts of activecompounds used in a given therapy will vary according to the specificcompound being utilized, the particular compositions formulated, themode of application, the particular site of administration, etc. Optimaladministration rates for a given protocol of administration can bereadily ascertained by those skilled in the art using conventionaldosage determination tests conducted with regard to the foregoingguidelines.

In general, compounds of the invention for treatment can be administeredto a subject in dosages used in prior tetracycline therapies. See, forexample, the Physicians' Desk Reference. For example, a suitableeffective dose of one or more compounds of the invention will be in therange of from 0.01 to 100 milligrams per kilogram of body weight ofrecipient per day, preferably in the range of from 0.1 to 50 milligramsper kilogram body weight of recipient per day, more preferably in therange of 1 to 20 milligrams per kilogram body weight of recipient perday. The desired dose is suitably administered once daily, or severalsub-doses, e.g., 2 to 5 sub-doses, are administered at appropriateintervals through the day, or other appropriate schedule.

It will also be understood that normal, conventionally known precautionswill be taken regarding the administration of tetracyclines generally toensure their efficacy under normal use circumstances. Especially whenemployed for therapeutic treatment of humans and animals in vivo, thepractitioner should take all sensible precautions to avoidconventionally known contradictions and toxic effects. Thus, theconventionally recognized adverse reactions of gastrointestinal distressand inflammations, the renal toxicity, hypersensitivity reactions,changes in blood, and impairment of absorption through aluminum,calcium, and magnesium ions should be duly considered in theconventional manner.

Furthermore, the invention also pertains to the use of a tetracyclinecompound of Formula I, II, III. Ilia, IV, IVa, V, Va, VI, VII, VIIa,VIII, VIIIa, IX, IXa, X, Xa, XI, Xa, XII, XIII, XIV, XV or XVI or acompound listed in Table 2, or any other compound described herein, forthe preparation of a medicament. The medicament may include apharmaceutically acceptable carrier and the tetracycline compound is aneffective amount, e.g., an effective amount to treat a tetracyclineresponsive state.

In one embodiment, the substituted tetracycline compound is a compoundof Table 2.

TABLE 2 Code Compound A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

AA

AB

AC

AD

AE

AF

AG

AH

AI

AJ

AK

AL

AM

AN

AO

AP

AQ

AR

AS

AT

AU

AV

AW

AX

AY

AZ

BA

BB

BC

BD

BE

BF

BG

BH

BI

BJ

BK

BL

BM

BN

BO

BP

BQ

BR

BS

BT

BU

BV

BW

BX

BY

BZ

CA

CB

CC

CD

CE

CF

CG

CH

CI

CJ

CK

CL

CM

CN

CO

CP

CQ

CR

CS

CT

CU

CV

CW

CX

CY

CZ

DA

DB

DC

DD

DE

DF

DG

DH

DI

DJ

DK

DL

DM

DN

DO

DP

DQ

DR

DS

DT

DU

DV

DW

DX

DY

DZ

EA

EB

EC

ED

EE

EF

EG

EH

EI

EJ

EK

EL

EM

EN

EO

EP

EQ

ER

ES

ET

EU

EV

EW

EX

EY

EZ

FA

FB

FC

FD

FE

FF

FG

FH

FI

FJ

FK

FL

FM

FN

FO

FP

FQ

FR

FS

FT

FU

FV

FW

FX

FY

FZ

GA

GB

GC

GD

GE

GF

GH

GI

GK

GL

GM

GN

GO

GP

GQ

GR

GS

GT

GU

GV

Example 1: Synthesis of Select Compounds of the Invention3-[3-((6aS,10S,10aS,11aR)-8-Carbamoyl-10-dimethylamino-4,6,6a,9-tetrahydroxy-5,7-dioxo-5,6a,7,10,10a,11,11a,12-octahydro-naphthacen-1-yl)-benzoylamino]-propionicacid ethyl ester (Compound AV)

An amount 1.00 g of 7-iodosancycline trifluoroacetic acid salt, 177 mgof palladium (0) tetrakistriphenylphosphine, 35 mg of palladium (II)acetate and 457 mg of ethyl 3-(3-borobenzoylamino)propionate, 98% wereloaded in a dry 20 mL microwave reaction vessel equipped with a magneticstir bar. Dry dimethylacetamide (DMA, 10 mL) was added and argon wasbubbled through the solution for 5 minutes. In a separate vial, sodiumacetate (487 mg) was dissolved in distilled water (5 mL) and argon wasbubbled through the solution for 5 minutes. The sodium acetate solutionwas added to the microwave reaction vessel which was sealed with acrimper. The reaction mixture was then subjected to microwaveirradiation for 10 minutes at 110° C., and the reaction was monitored byLC/MS. The reaction mixture was filtered through a pad of celite andwashed with methanol. After evaporation of organic solvents, the aqueoussolution was purified on a fluorinated DVB (DiVinylBenzene) column withgradients of a 50/50 methanol/acetonitrile, 0.1% TFA solution into a0.1% TFA water solution. The fractions were collected and evaporated toa minimum volume. The residue was then purified by preparative HPLCchromatography (C18, linear gradient 27-32% acetonitrile in water with0.2% formic acid). The fractions were evaporated and the resultingresidue was purified again by preparative HPLC chromatography (C18,linear gradient 20-35% acetonitrile in 20 mM aqueous triethanolamine, pH7.4) in order to separate the 4-epimers. The fractions were collectedand the organic solvent was evaporated. The resulting aqueous solutionwas loaded on a DVB column, washed with distilled water, and then with a0.1% hydrochloric acid solution. After eluting with a 50/50 mixture ofmethanol and acetonitrile, the solution was evaporated and the residuedried under high vacuum and P₂O₅ overnight to yield a yellow solid as anHCl salt. ESIMS: m/z 634 (MH+). ¹H-NMR (300 MHz, tetramethylsilane (TMS)as internal standard at 0 ppm): δ 7.78 (dm, 1H), 7.70 (m, 1H), 7.51 (t,1H), 7.45 (d, 2H), 6.92 (d, 1H), 4.13 (q, 2H), 4.00 (s, 1H), 3.63 (t,2H), 2.97-2.80 (m, 8H), 2.77 (dd, 1H), 2.64 (t, 2H), 2.52 (t, 1H),2.08-1.95 (m, 1H), 1.53 (q, 1H), 1.23 (t, 3H). Compounds AO, AP. AQ, AR,AS, AT, AU, AW, AX, AY, AZ, BA, BB, DK, DL, DM, DN, DO and DP wereprepared in a similar manner.

(4S,4aS,5aR,12aS)-7-[3-(2-Diethylamino-ethylcarbamoyl)-phenyl]-4-dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound BC)

An amount of 2.5 g of 7-iodosancycline trifluoroacetic acid salt, 221 mgof palladium (0) tetrakistriphenylphosphine, 43 mg of palladium (II)acetate and 777 mg of 3-carboxy-phenylboronic acid were loaded in a dry20 mL microwave reaction vessel equipped with a magnetic stir bar. DryDMA (13 mL) was added and argon was bubbled through the solution for 5minutes. In a separate vial, sodium acetate (105.99 g/mol, 1.215 g,11.46 mmol, 3.0 eq.) was dissolved in distilled water (7 mL) and argonwas bubbled through the solution for 5 minutes. The sodium acetatesolution was added to the microwave reaction vessel, which was sealedwith a crimper. The reaction mixture was then subjected to microwaveirradiation for 10 minutes at 110° C., and the reaction was monitored byLC/MS. The reaction mixture was filtered through a pad of celite andwashed with methanol. After evaporation of organic solvents, the aqueoussolution was purified on a fluorinated DVB (DiVinylBenzene) column withgradients of a 50/50 methanol/acetonitrile, 0.1% TFA solution into a0.1% TFA water solution. The fractions were collected and evaporated todryness to yield an orange solid, which was used in the next stepwithout further purification.

An amount of 340 mg of 7-(3-carboxy-phenyl)-sancycline TFA salt and 212mg of O-benzotriazol-1-yl-N,N,N′N′-tetramethyluroniumhexafluoro-phosphate were loaded in a dry 10 mL vial equipped with amagnetic stir bar. Dry DMA (2.5 mL) was added, followed bydiisopropylethylamine (180 μL). After 5 minutes of stirring at roomtemperature, N,N-diethyl-ethylenediamine, 98% (150 μL) was added, thereaction mixture was stirred at room temperature for 15 minutes and thereaction was monitored by LC/MS. The mixture was filtered throughcelite, evaporated in a rotary evaporator, and the residue was purifiedby preparative HPLC chromatography (C18, linear gradient 25-35%acetonitrile in water with 0.2% formic acid). The fractions werecombined, evaporated, and the resulting residue was purified again bypreparative HPLC chromatography (C18, linear gradient 20-35%acetonitrile in 20 mM aqueous triethanolamine, pH 7.4) in order toseparate the 4-epimers. The fractions were collected and the organicsolvent evaporated. The resulting aqueous solution was loaded on a DVBcolumn, washed with DI water, and then washed with a 0.1% hydrochloricacid solution. After eluting with a 50/50 mixture of methanol andacetonitrile, the solution was evaporated and the residue dried underhigh vacuum and P₂O₅ overnight to yield a yellow solid as an HCl salt.ESIMS: m/z 633 (MH+). ¹H-NMR (300 MHz, tetramethylsilane (TMS) asinternal standard at 0 ppm): δ 7.87 (dm, 1H), 7.79 (m, 1H), 7.60-7.47(m, 2H), 7.44 (d, 1H), 6.93 (d, 1H), 4.02 (s, 1H), 3.76 (t, 2H),3.45-3.30 (m, 6H), 3.02-2.85 (m, 8H), 2.78 (dd, 1H), 2.54 (t, 1H),2.10-1.95 (m, 1H), 1.53 (q, 1H), 1.35 (t, 6H). Compounds BD, BE, BF, BG,BH, BI, BJ, BK and DQ were prepared in a similar manner.

(4S,4aS,5aR,12aS)-4,7-Bis-dimethylamino-9-[3-(2-dimethylamino-ethylcarbamoyl)-phenyl]-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound BL)

An amount of 500 mg of 9-iodo-minocycline free base, 100 mg of palladium(0) tetrakis triphenylphosphine, 20 mg of palladium (II) acetate and 234mg of [3-(3-N,N-dimetylaminoetylaminocarbonyl)-phenyl]-boronic acid wereloaded in a dry 20 mL microwave reaction vessel equipped with a magneticstir bar. Dry DMA (4 mL) was added and argon is bubbled through thesolution for 5 minutes. In a separate vial, sodium acetate (274 mg) wasdissolved in DI water (2 mL) and argon was bubbled through the solutionfor 5 minutes. The sodium acetate solution was added to the microwavereaction vessel, which was sealed with a crimper. The reaction mixturewas then subjected to microwave irradiation for 10 minutes at 110° C.,and the reaction was monitored by LC/MS. The reaction mixture wasfiltered through a pad of celite and washed with methanol. Afterevaporation of organic solvents, the aqueous solution was purified on afluorinated DVB (DiVinylBenzene) column with gradients of a 50/50methanol/acetonitrile, 0.1% TFA solution into a 0.1% TFA water solution.The fractions were collected and evaporated to a minimum volume. Theresidue was then purified by HPLC chromatography (C18, linear gradient10-20% acetonitrile in water with 0.2% formic acid). The fractions werecombined, evaporated, and the resulting residue was purified again bypreparative HPLC chromatography (C18, linear gradient 10-20%acetonitrile in 20 mM aqueous triethanolamine, pH 7.4) in order toseparate the 4-epimers. The fractions were collected and the organicsolvent evaporated. The resulting aqueous solution was loaded on a DVBcolumn, washed with distilled water, and then with a 0.1% hydrochloricacid solution. After eluting with a 50/50 mixture of methanol andacetonitrile, the solution was evaporated and the residue dried underhigh vacuum and P₂O₅ overnight to yield a yellow solid as an HCl salt.ESIMS: m/z 648 (MH+). ¹H-NMR (300 MHz, tetramethylsilane (TMS) asinternal standard at 0 ppm): δ 8.26 (t, 1H), 8.16 (s, 1H), 7.94 (m, 2H),7.59 (t, 1H), 4.19 (s, 1H), 3.82 (t, 2H), 3.50-3.30 (m, 9H), 3.30-3.10(m, 2H), 3.10-2.90 (m, 9H), 2.62 (t, 1H), 2.42-2.30 (m, 1H), 1.71 (q,1H). Compound BM was prepared in a similar manner.

4aS,5aR,12aS)-7-[3-(2-Dimethylamino-ethylcarbamoyl)-phenyl]-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound BN)

An amount of 1.00 g of 7-iodo-4-dedimethyl-sancycline free base, 233 mgof palladium (0) tetrakis triphenylphosphine, 45 mg of palladium (II)acetate and 544 mg of[3-(3-N,N-dinmethylaminoethylaminocaibonyl)-phenyl]-boronic acid wereloaded in a dry 20 mL microwave reaction vessel equipped with a magneticstir bar. Dry DMA (8 mL) was added and argon was bubbled through thesolution for 5 minutes. In a separate vial, sodium acetate (640 mg) wasdissolved in distilled water (4 mL) and argon was bubbled through thesolution for 5 minutes. The sodium acetate solution was added to themicrowave reaction vessel, which was sealed with a crimper. The reactionmixture was then subjected to microwave irradiation for 10 minutes at110° C. and the reaction was monitored by LC/MS. The reaction mixturewas filtered through a pad of celite and washed with methanol. Afterevaporation of organic solvents, the aqueous solution was purified on afluorinated DVB (DiVinylBenzene) column with gradients of a 50/50methanol/acetonitrile, 0.1% TFA solution into a 0.1% TFA water solution.The fractions were collected and evaporated to a minimum volume. Theresidue was then purified by preparative HPLC chromatography (C18,linear gradient 20-35% acetonitrile in water with 0.2% formic acid). Thefractions were combined, evaporated, and the resulting residue waspurified again by preparative HPLC chromatography (C18, linear gradient15-35% acetonitrile in 20 mM aqueous triethanolamine, pH 7.4) in orderto separate the 4-epimers. The fractions were collected and the organicsolvent evaporated. The resulting aqueous solution was loaded on a DVBcolumn, washed with distilled water, and then with a 0.1% hydrochloricacid solution. After eluting with a 50/50 mixture of methanol andacetonitrile, the solution was evaporated and the residue dried underhigh vacuum and P₂O₅ overnight to yield a yellow solid as an HCl salt.ESIMS: m/z 562 (MH+). 1H-NMR (300 MHz, tetramethylsilane (TMS) asinternal standard at 0 ppm): δ 7.87 (din, 1H), 7.78 (s, 1H), 7.60-7.45(nm, 2H), 7.41 (d, 1H), 6.90 (d, 1H), 3.76 (m, 2H), 3.38 (t, 2H), 3.21(dd, 1H), 2.98 (s, 6H), 2.85-2.62 (m, 2H), 2.57-2.22 (m, 3H), 1.90-1.80(m, 1H), 1.48 (q, 1H).

(4S,4aS,5aR,12aS)-4-Dimethylamino-7-[3-(2-dimethylamino-acetylamino)-phenyl]-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound BO)

An amount of 2.50 g of 7-iodosancycline trifluoroacetic acid salt, 221mg palladium (0) tetrakis triphenylphosphine, 42 mg of palladium (II)acetate, and 812 mg of 3-amino-phenylboronic acid were loaded in a dry20 mL microwave reaction vessel equipped with a magnetic stir bar. DryDMA (13 mL) was added and argon was bubbled through the solution for 5minutes. In a separate vial, sodium acetate (1.22 g) was dissolved indistilled water (7 mL) and argon was bubbled through the solution for 5minutes. The sodium acetate solution was added to the microwave reactionvessel, which was sealed with a crimper. The reaction mixture was thensubjected to microwave irradiation for 20 minutes at 120° C., and thereaction was monitored by LC/MS. The reaction mixture was then filteredthrough a pad of celite and washed with methanol. After evaporation oforganic solvents, the aqueous solution was purified on a fluorinated DVB(DiVinylBenzene) column with gradients of a 50/50 methanol/acetonitrile.0.1% TFA solution into a 0.1% TFA water solution. The fractions werecollected and evaporated to dryness to yield a brown solid which is usedin the next step without further purification.

An amount of 250 mg of 7-(3-amino-phenyl)-sancycline TFA salt and 250 μLof diisopropylethylamine were loaded into a dry 5 mL microwave reactionvessel equipped with a magnetic stir bar. After 5 minutes of stirring,dimethylamino acetyl chloride, 85% (667 mg) was added, the reactionvessel was sealed, the reaction mixture was subjected to microwaveirradiation for 5 minutes at 100° C. and the reaction was monitored byLC/MS. The mixture was filtered through celite, evaporated in a rotaryevaporator, and the residue was purified by preparative HPLCchromatography (C18, linear gradient 10-30% acetonitrile in water with0.2% formic acid). The fractions were combined, evaporated, and theresulting residue was purified again by preparative HPLC chromatography(C18, linear gradient 15-25% acetonitrile in 20 mM aqueoustriethanolamine, pH 7.4) in order to separate the 4-epimers. Thefractions were collected and the organic solvent evaporated. Theresulting aqueous solution was loaded on a DVB column, washed withdistilled water, and then with a 0.1% hydrochloric acid solution. Aftereluting with a 50/50 mixture of methanol and acetonitrile, the solutionwas evaporated and the residue dried under high vacuum and P₂O₅overnight to yield a yellow solid as an HCl salt. ESIMS: m/z 591 (MH+).1H-NMR (300 MHz, tetramethylsilane (TMS) as internal standard at 0 ppm):δ 7.56 (m, 2H), 7.45-7.32 (m, 2H), 7.07 (d, 1H), 6.91 (d, 2H), 4.15 (s,2H), 4.04 (s, 1H), 3.20-2.70 (m, 15H), 2.48 (t, 1H), 2.04 (m, 1H), 1.51,(m, 1H). Compound BP was prepared in a similar manner.

(4S,4aS,5aR,12aS)-4-Dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy-methyl-amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound P)

A solution of 7-formylsanscycline TFA salt (2.23 g) andN,O-dimethylhydroxylamine hydrochloride (780 mg) inN,N-dimethylacetamide (15 mL) was stirred for 10 minutes at roomtemperature under argon atmosphere. To this solution was added sodiumcyanoborohydride (302 mg). The solution was stirred for 5 minutes andmonitored by LC-MS. The reaction mixture was poured into diethyl ether,and the resulting precipitates were collected by filtration undervacuum. The crude product was purified by preparative HPLC (C18 column,linear gradient 10-40% acetonitrile in 20 mM aqueous triethanolamine, pH7.4). The prep-HPLC fractions were collected, and the organic solvent(acetonitrile) was evaporated in vacuo. The resulting aqueous solutionwas loaded onto a clean PDVB SPE column, washed with distilled water,then with a 0.1 M sodium acetate solution followed by distilled water.The product was eluted with 0.1% TFA in acetonitrile. Afterconcentrating under vacuum, 565 mg was obtained as a TFA salt. The ‘T’FAsalt was converted to the hydrochloride salt by adding methanolic HCfollowed by in vacuo evaporation. This process was repeated twice togive a yellow solid. ESIMS: m/z 488 (MH+). ¹H NMR (300 MHz, CD₃OD δ 7.46(1H, J=8.6 Hz), 6.81 (d, 1H, J=8.6 Hz), 4.09 (d, 1H, J=1.0 Hz), 3.79 (d,1H, J=13.1 Hz), 3.73 (d, 1H, J=13.1 Hz), 3.36 (m, 1H), 3.27 (s, 3H),3.08-2.95 (8H), 2.61 (s, 3H), 2.38 (t, 1H, J=14.8), 2.22 (m, 1H), 1.64(m, 1H). Compounds Y, U and DR were prepared in a similar manner, andcompound DS may be synthesized in a similar manner.

(4S,4aS,5aR,12aS)-4-Dimethylamino-9-[(2,2-dimethyl-propylamino)-methyl]-7-(1-ethoxyimino-ethyl)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound N)

A solution of 7-acetyl-9-[(2,2-dimethylpropylamino)-methyl]-sancycline(0.383 mmol) and O-ethylhydroxylamine hydrochloride (2.30 mmol) inmethanol (15 mL) was stirred overnight. The solvent was evaporated andpurified by prep-HPLC (C18 column (linear gradient 15-30% acetonitrilein water with 0.1% TFA) to give a yellow solid. ESIMS: m/z 599 (MH+); ¹HNMR (300 MHz, CD₃OD) δ 7.60 (s, 1H), 4.33 (s, 2H), 5.06 (m, 2H), 4.16(2H, q, J=7.0 Hz), 4.08 (s, 1H), 3.11-2.90 (11H), 2.52 (m, 1H), 2.18 (m,1H), 2.15 (s, 3H), 1.28 (3H, t, J=7.0 Hz), 1.06 (s, 9H).

(4S,4aS,5aR,12aS)-9-Amino-7-(1-tert-butoxyimino-ethyl)-4-dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound 0)

A solution of 7-acetyl-9-aminosancycline hydrochloride (0.79 mmol) andO-tert-butylhydroxylamine hydrochloride (4.74 mmol) in methanol (10 mL)was stirred overnight. The methanol was evaporated and the resultingcompound purified by prep-HPLC using C18 column (linear gradient 15-35%acetonitrile in water with 0.1% TFA) to give a yellow solid: ESIMS m/z543 (MH+); ¹H NMR (300 MHz, CD₃OD) δ 7.54 (s, 1H), 4.14 (s, 1H),3.14-2.99 (9H), 2.52 (m, 1H), 2.20 (m, 1H), 2.16 (s, 3H), 1.32 (s, 9H).Compounds M and R were prepared in a similar manner and compound DU maybe prepared in a similar manner.

(4S,4aS,5aR,12aS)-9-Amino-4-dimethylamino-3,10,12,12a-tetrahydroxy-1,1,1-dioxo-7-[1-(2,2,2-trifluoro-ethoxyimino)-ethyl]-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound DT)

A solution of 7-acetyl-9-amino-sancycline (1.5 mmol) and2,2,2-trifluoroethylhydroxylamine hydrochloride (3 mmol) in methanol (20mL) was stirred overnight. The methanol was reduced and the crudeproduct was purified by prep-HPLC using C18 column (linear gradient10-35% acetonitrile in water with 0.1% TFA) to give a yellow solid: MS(Mz+1=569); ¹H NMR (300 MHz, CD₃OD) δ 7.48 (s, 1H), 4.63 (d, 1H, J=8.9Hz), 4.57 (d, 1H, J=8.9 Hz), 4.12, (d, 1H, J=0.9 Hz), 3.10-2.96 (9H),2.50 (m, 1H), 2.22 (s, 3H), 2.18 (m, 1H), 1.62 (m, 1H). Compounds M andR were prepared in a similar manner and compound DU may be prepared in asimilar manner.

(4S,4aS,5aR,12aS)-4-Dimethylamino-7-(ethoxyamino-methyl)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound AA)

An amount of 7-formyl-sancycline (1.5 g) was combined with methanol (30mL) and O-(ethoxy)methylamine (1.5 g). The reaction solution was stirredat room temperature for 3 hours under a blanket of argon and wasmonitored by HPLC and LC/MS. The solvent was evaporated in vacuo and ayellow solid (2.3 g) was isolated as an oxime. ESIMS: m/z 485 (MH+).

The oxime (2.3 g) was suspended in methanol saturated with HCl (45 mL)and cooled in an ice bath. An amount of 585 mg of NaCNBH₃ was added insmall batches followed by a few drops of methanol saturated with HCl viasyringe. The reducing agent was added over the course of 2 hours and thereaction was monitored by HPLC and LC/MS. The solvent was evaporated invacuo and was purified. The compound purified by HPLC (C18, lineargradient 10-45% acetonitrile in 20 mM aqueous triethanolamine, pH 7.4).The purified compound was dried in vacuo and redissolved in methanol (20mL) saturated with HCl to exchange the salt. The compound was driedovernight over P₂O₅ to yield the product (0.21 mg, 13%) as a yellowpowder. ESIMS: m/z 488 (MH+). ¹H NMR (300 MHz, CD₃OD) δ 7.63 (1H, d, J=9Hz), 6.93 (1H, d, J=9 Hz), 4.53 (s, 1H), 4.17 (m, 3H), 3.25 (m, 1H),3.07 (m, 8H), 2.44 (m, 1H), 2.31 (m, 1H), 1.62 (m, 1H), 1.29 (3H, t, J=7Hz). Compounds AM, AB, AE, AF and AG were prepared in a similar manner.

(4S,4aS,5aR,12aS)-4-Dimethylamino-3,10,12,12a-tetrahydroxy-7-(isopropoxyamino-methyl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound DV)

A solution of 7-formylsancycline (1.8 mmol) and O-isopropylhydroxylaminehydrochloride (9 mmol) in methanol (25 mL) was stirred overnight. Thesolvent was reduced and the crude product was used for the next reactionwithout further purification. A solution of7-(isopropoxyimino-methyl)-sancycline (2 mmol) in methanol saturatedwith HCl was cooled in an ice-bath and NaCNBH₃ was added portion-wisewhile stirring at the same temperature. The solvent was evaporated andthe crude product was purified by prep-HPLC using C18 column (lineargradient 15-30% acetonitrile in 20 mM aqueous triethanolamine, pH 7.4)to give a yellow solid: MS (Mz+1=502); ¹H NMR (300 MHz, CD₃OD) δ 7.63(d, 1H, J=8.7 Hz), 6.92 (d, 1H, J=8.7 Hz), 4.44 (m, 1H), 4.14 (d, 1H,J=1.2 Hz), 3.27-2.97 (9H), 2.43 (t, 1H, J=14.4), 2.27 (m, 1H), 1.29 (m,6H). Compounds AM. AB, AE, AF, AG, DX, DZ, EA. EB and ED were preparedin a similar manner and compounds EE and EF may be synthesized in asimilar manner.

(4S,4aS,5aR,12aS)-4-Dimethylamino-7-[(2-fluoro-ethoxyamino)-methyl]-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound DW)

A solution of 7-formylsancyline (4 mmol) and 2-fluoroethylhydroxylaminehydrochloride (10 mmol) in methanol (50 mL) was stirred overnight, afterwhich LC-MS showed completion of the reaction. The solvent was reducedand the crude product was used for the next reaction without furtherpurification. To a cooled solution of7-(2′-fluoro-ethoxyimino-methyl)-sancycline (2 mmol) in methanolsaturated with HCl was added portion-wise NaCNBH₃ (8 mmol) over 8 hourswhile stirring at the same temperature. The solvent was reduced and thecrude product was purified by prep-HPLC using C18 column (lineargradient 10-40% acetonitrile in 20 mM aqueous triethanolamine, pH 7.4)to give a yellow solid: MS (Mz+1=506); ¹H NMR (300 MHz, CD₃OD) δ 7.65(d, 1H, J=8.8 Hz), 6.93 (d, 1H, J=8.8 Hz), 4.75 (m, 1H), 4.61-4.55 (3H),4.46 (m, 1H), 4.36 (m, 1H), 4.16 (d, 1H, J=1.2 Hz), 3.26-2.97 (9H), 2.45(t, 1H, J=14.4), 2.31 (m, 1H), 1.63 (m, 1H). Compounds AM, AB, AE, AF,AG, DX, DZ. EA, EB and ED were prepared in a similar manner andcompounds EE and EF may be synthesized in a similar manner.

4S,4aS,5aR,12aS)-4-Dimethylamino-3,10,12,12a-tetrahydroxy-7-(3-imino-isoxazolidin-2-ylmethyl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound EC)

A solution of 7-formylsancycline (2 mmol) and 3-aminooxy-propionnitrile(4 mmol) in methanol (30 mL) was stirred overnight. The solvent wasreduced and the crude product was used for the next reaction withoutfurther purification. A solution of7-(2′-cyanoethoxyimmuno-methyl)-sancycline in methanol and HCl wascooled with an ice-bath and NaCNBH₃ was added portion-wise and stirredfor 1.5 hours. The solvent was evaporated and the compound was purifiedby prep-HPLC using C-18 column (linear gradient 10-40% acetonitrile in20 mM aqueous triethanolamine, pH 7.4) to give a yellow solid: MS(Mz+1=513); ¹H NMR (300 MHz, CD₃OD) δ 7.47 (d, 1H, J=8.8 Hz), 6.86 (d,1H, J=8.8 Hz), 5.11 (d, 1H, J=15.9 Hz), 4.96 (d, 1H, J=15.9 Hz), 4.41(m, 2H), 4.11 (s, 1H), 3.50 (t, 2H, J=8.4 Hz), 3.20-2.94 (9H), 2.38 (t,1H, J=15.3 Hz), 2.28 (m, 1H), 1.60 (m, 1H). Compounds AM, AB, AE, AF,AG, DX, DZ, EA, EB and ED were prepared in a similar manner andcompounds EE and EF may be synthesized in a similar manner.

(4S,4aS,5aR,12aS)-4-Dimethylamino-3,12,12a-trihydroxy-1,11-dioxo-7-pyrazin-2-yl-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound W)

An amount of 7-iodo-sancycline (1 g) was combined with CuI (0.029 g),Pd₂(dba)₃ (0.140 g), tri-2-furylphosphine (0.284 g),2-tributylstannylpyrazine (0.677 g,), and DMF (6 mL) in a 20 mL glassmicrowave vial. The reaction mixture was subjected to microwaveirradiation for 10 minutes at 100° C. on high absorbance, and wasmonitored by HPLC and LC/MS. The solvent was evaporated in vacuo and thefree-base of the above compound was made by pouring 8 g of product into1.8 L water (0.1% TFA). Celite was added, and material was filteredthrough a celite plug. The water filtrate was loaded onto a prepared DVBcolumn, and washed with water (0.1% TFA), 0.25 M NaOAc until a basic pHwas obtained. The DVB column was then washed with distilled water untila neutral pH was obtained, and the compound was then eluted as thefree-base with CH₃CN.

The resulting 7-pyrazine-sancycline-free base (1 g) was combined withdry THF (15 mL) and the reaction solution was cooled in an ice bathunder a blanket of argon. Potassium r-butoxide (1.17 g) was added in oneaddition. The resulting heterogeneous mixture was stirred in an ice bathfor 45 minutes. An amount of N-phenylbis(trifluommethanesulfonamide)(1.49 g) was added in one addition. The resulting solution was stirredin an ice bath for 45 minutes, then was warmed to room temperature andstirred another 1 hour. The reaction monitored by HPLC and LC/MS. Thereaction mixture was poured into 200 mL 0.5M HCl, celite was added, andmixture was filtered through a celite plug. The water filtrate wasloaded onto a prepared DVB column, washed with 0.5M HCl, water, theneluted with CH₃CN (0.1% TFA). The product was evaporated to dryness andpurified by HPLC (C18, linear gradient 5-45% acetonitrile in water with0.1% TFA). Clean fractions were evaporated to dryness.

The resulting 7-pyrazine-10-triflate-sancycline (0.220 g) was combinedwith ammonium formate (0.112 g), lithium chloride (0.074 g),Pd₂(dppf)₂Cl₂ (0.052 g) DMA (1.5 mL) and water (1.5 mL) in a glassmicrowave vial, which was then purged with argon and microwaveirradiated for 10 minutes at 100° C. on high absorbance. The reactionwas monitored by HPLC and LC/MS and was poured into 100 mL water (0.1%TFA), and filtered through celite. The yellow eluent was loaded onto aprepared 2 g DVB cartridge and eluted with CH₃CN (0.1% TFA). The solventwas evaporated and purified by HPLC (C18, linear gradient 5-45%acetonitrile in water with 0.1% TFA). The purified compound was dried invacuo, redissolved in methanol (20 mL) saturated with HCl and driedovernight over P₂O₅ to yield the product (0.035 g, 16%) as a yellowpowder. ESIMS: m/z 477 (MH+). ¹H NMR (300 MHz, CD₃OD) δ 8.77 (m, 1H),8.71 (m, 1H), 8.63 (m, 1H), 8.14 (m, 1H), 7.71 (m, 1H), 7.57 (m, 1H),3.99 (m, 1H), 2.97 (m, 9H), 2.63 (m, 1H), 2.04 (m, 1H), 1.62 (m, 1H).Compounds D, E, F, G and S were prepared in a similar manner.

(4S,4aS,5aR,12aS)-4-Dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-7-(1H-pyrrol-2-yl)-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound T)

An amount of 7-iodo-sancycline (1 g) was combined with Pd(OAc)₂ (0.034g), methanol (1 mL), and DMF (2 mL) in a glass microwave vial and thereaction mixture was purged with argon. An amount of Na₂CO₃ (0.482 g)was dissolved in water (I mL) and was added to the reaction vessel. Anamount of 1-N-Boc-pyrrole-2-boronic acid (0.645 g) was dissolved in DMF(1 mL) and added to the reaction vessel. The resulting mixture wasmicrowave irradiated for 10 minutes at 100° C. and the reaction wasmonitored by HPLC and LC/MS. The reaction mixture was filtered throughcelite, and solvent was reduced in vacuo. The crude reaction mixture wasthen precipitated in 500 mL diethyl ether to yield a yellow precipitate,which was then filtered, rinsed with fresh diethyl ether, and driedunder vacuum, to yield 700 mg of a yellow solid. The yellow solid wasadded to TFA (10 mL) and stirred at room temperature 5 minutes, followedby evaporation of the solvent. The resulting material was purified byHPLC (C18, linear gradient 15-50% acetonitrile in water with 0.1% TFA),dried in vacuo, redissolved in methanol (20 mL) saturated with HCl anddried overnight over P₂O₅ to yield the product (0.020 g, 3%) as a yellowpowder. ESIMS: m/z 480 (MH+). ¹H NMR (300 MHz, CD₃OD) δ 7.53 (1H, d,J=9H z), 6.87 (1H, d, J=9 Hz), 6.80 (m, 1H), 6.16 (m, 1H), 6.08 (m, 1H),4.06 (s, 1H), 3.18 (m, 1H), 2.98 (m, 9H), 2.49 (m, 1H), 2.09 (m, 1H),1.61 (m, 1H). Compounds J, K and L were prepared in a similar manner.

(4R,4aS,5aR,12aS)-4-Dimethylamino-7-(3-dimethylamino-1-ethoxyimino-propyl)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound AN

A solution of 7-(3′-dimethylamino-propionyl)-sancycline (5.12 mmol) and0-ethoxylamine hydrochloride (41 mmol) in N,N-dimethylacetamide wasstirred at 80° C. under microwave conditions for 70 minutes. The productwas purified by prep-HPLC using a C18 column (linear gradient 10-40%acetonitrile in water with 0.1% TFA) to give a yellow solid: ESIMS: m/z557 (MH+); ¹H NMR (300 MHz, CD₃OD) δ 7.39 (m, 1H), 6.91 (m, 1H), 4.86(1H, d, J=3.9 Hz), 4.26-4.08 (m, 2H), 3.5 (m, 1H), 3.30-2.87 (18H), 2.50(m, 1H), 2.20 (m, 1H), 1.56 (m, 1H), 1.36-1.19 (m, 3H). Compound O wasalso prepared in this manner.

(4S,4aS,5aR,12aS)-4-Dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-7-(pyrrol-1-yliminomethyl)-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound AC)

An amount of 7-formyl sancycline (0.4 g) was combined with 1-aminopyrrole (0.223 g) and DMA (8 mL) and was stirred at room temperatureunder a blanket of argon for 30 minutes. The reaction was monitored byHPLC and LC/MS. The crude reaction mixture was poured into water (0.1%TFA) (100 mL) and loaded onto a prepared 5 g DVB cartridge. The loadedcartridge was washed with water, then eluted with CH₃CN (0.1% TFA) andthe product was purified by HPLC (C18, linear gradient 10-70%acetonitrile in 20 mM aqueous triethanolamine, pH 7.4). The purifiedcompound was dried in vacuo, redissolved in methanol (20 mL) saturatedwith HCl and was dried overnight over P₂O₅ to yield the product (0.035g, 8%) as a yellow powder. ESIMS: m/z 507 (MH+). ¹H NMR (300 MHz, CD₃OD)δ 8.78 (s, 1H), 8.12 (1H, d, J=9 Hz), 7.23 (2H, t, J=3 Hz), 6.93 (1H, d,J=9 Hz), 6.17 (2H, t, J=3 Hz), 4.08 (s, 1H), 3.54 (m, 1H), 2.97 (m, 9H),2.47 (m, 1H), 2.24 (m, 1H), 1.65 (m, 1H). Compound X was also preparedin this manner.

(4S,4aS,5aR,12aS)-7-(N,N″-Diethyl-hydrazinomethyl)-4-dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound Z)

An amount of 7-formyl sancycline (0.5 g) was combined with1,2-diethylhydrazine (0.546 g), triethylamine (0.472 g) and DMA (10 mL)and was stirred at room temperature under a blanket of argon 45 minutes.An amount of NaCNBH₃ (0.084 g) was added to reaction mixture and stirredovernight at room temperature. Reaction monitored by HPLC and LC/MS andcomplete by morning. Poured reaction mixture into water (0.1% TFA),loaded onto a prepared 5 g DVB cartridge, and eluted with CH₃CN (0.1%TFA). Compound purified by HPLC (C18, linear gradient 5-60% acetonitrilein 20 mM aqueous triethanolamine, pH 7.4). Purified compound was driedin vacuo and redissolved in methanol (20 mL) saturated with HCl toexchange the salt. The compound was dried overnight over P₂O₅ to yieldthe product (0.030 g, 6%) as a yellow powder. ESIMS: m/z 515 (MH+). ¹HNMR (300 MHz, CD₃OD) δ 7.53 (1H, d, J=9 Hz), 6.87 (1H, d, J=9 Hz), 4.18(m, 1H), 4.06 (s, 2H), 3.19 (m, 1H), 3.00 (m, 10H), 2.40 (m, 1H), 2.20(m, 1H), 1.64 (m, 1H), 1.24 (3H, t, J=9 Hz), 1.13 (m, 3H).

Allyl-((6aS,10S,10aS,11aR)-8-carbamoyl-10-dimethylamino-4,6,6a,9-tetrahydroxy-5,7-dioxo-5,6a,7,10,10a,11,11a,12-octahydro-naphthacen-1-ylmethyl)-carbamicacid methyl ester (Compound F)

A solution of 7-formylsancycline (1.5 mmol) and allylamine (4.5 mmol) in1,2-dichloroethane (50 mL) was stirred for 30 minutes. Sodiumtriacetoxyborohydride was added and stirred for additional 3 hours. Thesolvent and excess reagent was evaporated and the crude material waspurified by prep-HPLC using C18 column (linear gradient 15-30acetonitrile in water with 0.2% formic acid) to give7-allylaminomethyl-sancycline as a yellow solid: ESIMS: m/z 484 (MH+).

To a solution of 7-allylaminomethyl-sancycline (0.78 mmol) inN,N-dimethylacetamide (7 mL) was added methylchloroformate (1.6 mmol)dropwise and the reaction mixture was stirred for 1 hour. An additionalamount of methylchloroformate (1.6 mmol) was added and stirred foradditional 3 hours. The resulting product was purified by prep prep-HPLCusing C18 column (linear gradient 15-30 acetonitrile in water with 0.2%formic acid) to give a yellow solid: ESIMS: m/z 542 (MH+); ¹H NMR (300MHz, CD₃OD) δ 7.34 (1H, d, J=8.5 Hz), 6.82 (0H, d, J=8.5 Hz), 5.71 (m,1H), 5.06 (m, 2H), 4.47 (m, 2H), 4.08 (1H, d, J=0.9 Hz), 3.84-3.65 (m,2H), 3.71 (s, 3H), 3.21-2.92 (9H), 2.30-1.94 (2H), 1.59 (m, 1H).

(4S,4aS,5aR,12aS)-4,7-Bis-dimethylamino-3,10,12,12a-tetrahydroxy-9-(methoxyimino-methyl)-1,11-dioxo-1.4.4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound V)

A solution of 9-formylminocycline (1.19 mmol) and O-methylhydroxylaminehydrochloride (5.96 mmol) in methanol (15 mL) was stirred for 1.5 hours.The solvent was evaporated and purified by prep-HPLC using C18 column(linear gradient 10-50% acetonitrile in 20 mM aqueous triethanolamine,pH 7.4) to give a yellow solid: ESIMS m/z 515 (MH+); ¹H NMR (300 MHz,CD₃OD) δ 8.42 (s, 1H), 8.09 (s, 1H), 4.13 (1H, d, J=1.2 Hz), 3.99 (s,3H), 3.35 (m, 1H), 3.09-2.98 (14H), 2.43 (m, 1H), 2.24 (m, 1H), 1.69 (m,1H). Compounds AK and AH may be prepared as described above.

(4,S,4aS,5aR,12aS)-4-Dimethylamino-3,10,12,12a-tetrahydroxy-7-(2-methylamino-3,4-dioxo-cyclobut-1-enyl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound AD

A mixture of 7-iodosancycline (2 mmol),3-isopropooxy-4-tributylstannanyl-cyclobut-3-ene-1,2-dione (4.4 mmol),tetrakis(triphenylphosphine)palladium (0.4 mmol) and CuI (0.4 mmol) inN,N-dimethylacetamide was microwave irradiated for 50 minutes at 80° C.The resulting compound was purified using a DVB column to give7-(2′-isopropoxy-3′,4′-dioxo-cyclobut-1′-enyl)-sancycline as a yellowsolid::ESIMS m/z 553 (MH+).

To a solution of7-(2′-isopropoxy-3′,4′-dioxo-cyclobut-1′-enyl)-sancycline (0.9 mmol) inmethanol (20 mL) was added 1 mL of 33% methylamine in absolute ethanoland the reaction mixture was stirred for 40 minutes. The resultingproduct was purified prep-HPLC using C18 column (linear gradient 10-40%acetonitrile in 20 mM aqueous triethanolamine, pH 7.4) to give a yellowsolid: ESIMS m/z 524 (MH+); ¹H NMR (300 MHz, CD₃OD) δ 7.46 (1H, d, J=8.7Hz), 6.88 (1H, d, J=8.7 Hz), 4.01 (s, 1H), 3.27 (s, 3H), 3.07-2.82 (9H),2.45 (m, 1H), 2.10 (m, 1H), 1.52 (m, 1H). Compounds AI and AJ may beprepared in this manner.

(4S,4aS,5aR,12aS)-4-Dimethylamino-7-{[(2-ethoxyimino-propyl)-methyl-amino]-methyl}-3,10,12,12a-tetrahydroxy-1,11-dioxo-1.4.4a,5.5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound AL)

A solution of 7-formylsancycline (2 mmol) andmethyl-(2-methyl-[1,3]dioxolan-2-ylmethyl)-anine (6 mmol) inN,N-dimethylformamide (30 mL) was stirred for 40 minutes. Sodiumtriacetoxyborohydride (6 mmol) was added and the reaction was stirredfor 6 hours. The solvent was evaporated and the crude material wasdissolved in a mixture of tetrahydrofuran (10 mL), acetic acid (10 mL)and 6M HCl (10 mL). This solution was stirred at 60° C. for 6 hours.Upon completion, the solvent and excess reagents were evaporated and thecrude material was purified by prep-HPLC using C18 column (lineargradient 20-50% acetonitrile in 20 mM aqueous triethanolamine, pH 7.4)to give 7-{[(methyl-2′-oxo-propyl)-methyl-amino]-methyl}-sancycline as ayellow solid: ESIMS m/z 514 (MH+).

A solution of 7-{[methyl-(2′-oxo-propyl)-amino]-methyl}-sancycline (0.63mmol) and O-ethylhydroxyamine hydrochloride (3.15 mmol) in methanol (15mL) was stirred for 8 hours. The solvent was evaporated and purified byprep-HPLC using C18 column (linear gradient 20-50% acetonitrile in 20 mMaqueous triethanolamine, pH 7.4) to give a yellow solid: ESIMS m/z 557(MH+); ¹H NMR (300 MHz, CD₃OD) δ 7.69 (1H, d, J=8.7 Hz), 6.99 (1H, d,J=8.7 Hz), 4.65 (m, 1H), 4.35 (m, 1H), 4.24 (2H, q, J=7.1 Hz), 4.15 (s,1H), 4.07 (brs, 2H), 3.24-2.85 (12H), 2.50 (m, 1H), 2.30 (m, 1H), 1.95(s, 3H), 1.52 (m, 1H), 1.31 (3H, t, J=7.1 Hz).

(4aS,5aR,12aS)-7-Dimethylanino-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2,9-dicarboxylicacid 2-amide 9-(hydroxy-methyl-amide)4-dedimethylamino-minocycline-9-N-methylhydroxamic acid (Compound CC)

Minocycline/HCl salt (200 g, 0.406 mol) was suspended in 3 L water andan amount of NaHCO₃ (34 g, 0.406 mol) was added in 3 portions and the pHwas adjusted to 6.5-7.0. The solution was then extracted with 2×1.5 LCH₂Cl₂. The solution was concentrated to dryness to give minocycline asthe freebase, then redissolved in THF (1.6 L) and was charged in a 3 L3-necked flask equipped with an over-head stirrer and a temperatureprobe while under argon. An amount of methyl iodide (289 g, 2.03 mol)was added and the solution was heated at 40-45° C. for approximately 16hours, at which point it was verified by HPLC that no minocycline wasleft in solution. The solution was then poured into 6 L of heptane whileon ice bath and stirred for at least 20 minutes at <5° C. Theprecipitate was filtered and washed with hexane (400 mL). The solid wasdried under reduced pressure to a constant weight. An amount of 186 gmethylammonium salt of minocycline was isolated.

In a 3 L, 3-necked RBF equipped with overhead stirrer and a temperatureprobe a mixture of 200 ml DMF, 50 ml TFA, and 15 ml water was cooled onan ice bath to <5° C. An amount of 4-methylammonium minocycline (100 g)was added to the flask. Upon dissolution, Zn powder (14 g, 100 mesh) wasadded in 6 portions approximately every 30 minutes (˜2.33 g eachaddition). The reaction was monitored by HPLC. When less thanapproximately 10% of 4-trimethyammonium minocycline remained, thesolution was filtered through a bed of Celite and was washed with 500 mLwater. The solution was then poured into 2 L of water and the pH wasadjusted with aqueous ammonia to a pH of 3.5. The aqueous solution wasextracted first with 1 L dichloromethane (two times) and the combinedorganic layers were back-washed with 1 L water, dried on sodium sulfate,filtered and concentrated under reduced pressure down to an oil, toyield 4-dedimethylamino minocycline (48 g).

An amount of 4-dedimethyl minocycline (48 g, 0.115 mol) was charged in aflask under argon atmosphere and methanesulfonic acid (350 mL) wasadded. Ag₂SO₄ (75 g, 0.24 mol) and iodine (61.5 g, 0.24 mol) were addedand the mixture was stirred for 3 hours. Upon HPLC confirming completionof the reaction, the mixture was poured into 4% aqueous sodium sulfite(3.5 L) and was stirred for at least one hour. The solution was filteredthrough a bed of Celite, then washed with 200 ml of water. The aqueouslayer was loaded onto a column containing divinylbenzyl resin. Agradient of 20-60% organic (1:1 methanol:acetonitrile) in water with anoverall trifluoroacetic acid of 1.0% was used to elute compound4-dedimethylamine-9-iodo minocycline. The combined fractions werereduced of organic, the pH adjusted with aqueous NaHCO₃ to a pH of 7 andextracted with methylene chloride to give 20 g of4-dedimethylamine-9-iodo minocycline as the freebase.

To a 500 mL flask was added (2.00 g) 4-dedimethylamino-9-iodominocycline free base and NMP (37 mL), N-hydroxysuccinimide (3.9 g). Toremove residual water from the above reactants, toluene was added (37mL), the flask was placed on the rotary evaporator (35 mm Hg, 45° C.)until all the toluene was evaporated. The flask was backfilled withargon and the contents were then transferred via cannula to a dry 500 Lflask. To the 500 mL flask was addedtetrakis-(triphenylphosphine)palladium(0) (2.00 g) and DIEA (2.60 mL).The flask was placed under vacuum (20 mm Hg) and purged 3 times withcarbon monoxide. The flask was then heated to 60° C. under 1.0 atm ofcarbon monoxide and let stir for 1 hour. Subsequently,methylhydroxylamine (1.7 mL) and DIEA (0.5 mL) was added and thereaction was heated in a microwave reactor for 1 minute at 100° C. Thereaction was added to water (1.0 L) and the pH was lowered to 2 usingtrifluoroacetic acid. The solution was then filtered through celite,loaded onto a reverse phase column and the crude product was purified byreverse phase HPLC (C18, linear gradient 10-30% MeCN in water with 0.1%TFA). The fractions containing the final product were loaded onto DVBplug, washed with aqueous HCl (1.0 L, 0.01 N) and eluted withacetonitrile to give the HCl salt of4-dedimethylamino-9-N-methylhydroxamic acid minocycline (1000 mg, 44%).¹H-NMR (300 MHz, chemical shifts in ppm with TMS as internal referenceat 0 ppm) δ 1.5-1.7 (m, 1H), 2.1-2.25 (m, 1H), 2.35-2.7 (m, 3H), 2.9-3.1(m, 1H), 3.2-3.3 (brs, 7H), 3.35 (s, 2H), 3.45 (s, 2H), 7.91 (s, 1H). MWcalcd for C₂₃H₂₅N₃O₉ 487.46, ESIMS obs. m/z 488.25 (MH+). Compounds BQ,BR, BS, BT, BU, BV, BW, BX, BY, BZ, CA, CB, CD, CE, CF, EJ, EK and EMweir prepared as described above.

(4aS,5aR,12aS)-7-Dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2,9-dicarboxylicacid 2-amide 9-tert-butylamide (Compound EI)

To a 500 mL flask was added (2.00 g, 4.30 mmol) 4-dedimethylamine-9-iodominocycline free base (see synthesis of compound CC), NMP (37 mL).N-hydroxysuccinimide (3.9 g, 38 mmol). To remove residual water from theabove reactants toluene was added (37 mL), the flask was placed on therotary evaporator (5 mm Hg, 45° C.) until all the toluene wasevaporated. The flask was backfilled with argon and the contents werethen transferred via cannula to a dry 500 L flask. To the 0.5 L flaskwas added tetrakis(triphenylphosphine)palladium(0) (2.00 g, 1.67 mmol)and DIEA (2.60 mL, 1.48 mmol). The flask was placed under vacuum (20 mmHg) and purged 3 times with carbon monoxide. The flask was then heatedto 60° C. under 1.0 atm of carbon monoxide and let stir for 1 hour untilall 4-dedimethlyamino-9-iodo minocycline was consumed and a peak for thecorresponding NHS-ester intermediate (M+1) of 556 M/Z was formed asdetermined via LCMS. Subsequently, tert-butylamine (4.0 mL, 38 mmol) andDIEA (4.0 mL, 38 mmol) was added and the reaction was heated in amicrowave reactor for 1 minute at 100 C. The reaction was added toacetonitrile (150 mL) followed by water (0.8 L) and the pH was loweredto pH 2 using trifluoroacetic acid. The solution was then filteredthrough celite to remove the catalyst, loaded onto a reverse phasecolumn and the crude product was purified by HPLC (C18, linear gradient30-45% acetonitrile in water with 0.2% Formic acid). The fractionscontaining the final product were loaded onto DVB plug, washed withaqueous HCl (1.0 L, 0.01 N) and eluted with methanol to give the HClsalt of 4-dedimethyl-9-tertbutylcarboxamido minocycline (500 mg, 0.91mmol, 20%). ¹H-NMR (300 MHz, chemical shifts in ppm with TMS as internalreference at 0 ppm) δ 1.4 (s, 9H), 1.6-1.8 (m, 1H), 2.1-2.25 (m, 1H),2.35-2.7 (m, 3H), 2.9-3.1 (m, 1H), 3.15-3.3 (m, 1H), 3.38 (s, 1H), 8.45(s, 1H). MS (electron spray) calcd for CH₂₆H₃₁N₃O₈ 513.54, found (MH⁺)514.25. Compounds BQ, BR, BS, BT, BU, BV, BW, BX, BY, BZ, CA, CB, CD,CE, CF, EJ, EK and EM were prepared as described above.

(5aR,6aS,10aS)-9-Carbamoyl-4-dimethylamino-1,8,10a,11-tetrahydroxy-10,12-dioxo-5,5a,6,6a,7,10,10a,12-octahydro-naphthacene-2-carboxylicacid 2,2-dimethyl-propyl ester (Compound EL)

To a 250 mL flask was added (1.50 g, 2.78 mmol) 4-dedimethylamine-9-iodominocycline free base (see synthesis of compound CC) and NMP (12 mL). Toremove residual water from the above reactants, toluene was added (25mL), the flask was placed on the rotary evaporator (5 mm Hg, 45° C.)until all the toluene was evaporated. The flask was backfilled withargon and the contents were then transferred via cannula to a dry 50 mLflask. To the 50 mL flask was addedtetrakis(triphenylphosphine)palladium(0) (0.70 g, 0.56 mmol) and DIEA(1.30 mL, 8.33 mmol). The flask was placed under vacuum (30 mm Hg) andpurged 3 times with carbon monoxide. The flask was then heated to 60° C.under 1.0 atm of carbon monoxide. An amount of neopentyl alcohol (7.5mL, 68 mmol) was added and the reaction was stirred overnight until all4-dedimethlyamino-9-iodo minocycline was consumed and the product wasformed as determined via LCMS. The reaction was added to acetonitrile(300 mL) followed by the addition of water (0.7 L) and lastly the pH waslowered to pH 2 using trifluoroacetic acid. The solution was thenfiltered through celite to remove the catalyst, loaded onto a reversephase column and the crude product was purified by HPLC (C18, lineargradient 50-60% acetonitrile in water with 0.2% Formic acid). Thefractions containing the final product were loaded onto DVB plug, washedwith aqueous HCl (1.0 L, 0.01 N) and eluted with methanol to give theHCl salt of 4-dedimethyl-9-neopentylcarboxyester minocycline (450 mg,0.97 mmol, 35%). ¹H-NMR (300 MHz, chemical shifts in ppm with TMS asinternal reference at 0 ppm) δ 1.08 (s, 9H), 1.6-1.8 (m, 1H), 2.1-2.25(m, 1H), 2.35-2.7 (m, 3H), 2.9-3.15 (m, 1H), 3.15-3.0 (m, 1H), 4.05-4.2(m, 2H), 8.4 (s, 1H). MS (electron spray) calcd for C₂₇H₃₂N₂O₉ 528.54,found (M+1) 529.25. Compounds BQ, BR, BS, BT, BU, BV, BW, BX, BY, BZ,CA, CB, CD, CE, CF, EJ, EK and EM were prepared as described above.

(4S,4aS,5aR,12aS)-4,7-Bis-dimethylamino-9-(1-ethoxyimino-ethyl)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound CG)

A 100 mL round-bottom flask was charged with a stir bar,9-acetylminocycline (1.75 g), O-ethylhydroxylamine (1.548 g), and 30 mLof methanol. The reaction mixture was stirred at room temperature for 3hours, while the reaction progress was monitored with HPLC/LCMS. Uponcompletion, the reaction mixture was poured in water (500 mL) andpurified by preparative HPLC (C18, linear gradient 15-55% acetonitrilein 20 mM aqueous triethanolamine and TFA, pH 7.4). The product fractionswere diluted with water, loaded onto a DVB plug (0.5″×3″ diam), andwashed thoroughly with water. The product was then eluted with methanoland the solution was evaporated to dryness. An amount of 0.454 g ofcompound was isolated. ESIMS: m/z 543.4 (MH+) obs. ¹H NMR (300 MHz,CD₃OD) δ 7.34 (s, 1H), 4.18 (q, 2H), 3.68 (t, 1H), 3.36 (m, 1H), 2.90(m, 2H), 2.87 (s, 6H), 2.59 (s, 6H), 2.20 (s, 3H), 2.19 (m, 2H), 2.61(q, 1H), 1.30 (t, 3H).

(4aS,5aR,12aS)-7-Dimethylamino-3,10,12,12a-tetrahydroxy-9-(1-methoxyimino-ethyl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound CH)

To a mixture of 9-acetyl-4-dedimethylaminominocycline (2.02 g) andMeONH₂ (1.56 g) was added methanol (30 mL) and the reaction was stirredat room temperature for 2.4 hours. Upon completion of the reaction(monitored via LCMS), the reaction mixture was poured into water and waspurified by preparative HPLC (C18, linear gradient 15-55% acetonitrilein 20 mM aqueous triethanolamine and TFA, pH 7.4). Isolated 0.667 g.ESIMS m/z 486.25 (MH+) ¹H NMR (300 MHz, CD₃OD) δ 7.34 (s, 1H), 3.93 (s,3H), 3.38 (dd, 1H), 3.26 (m, 1H), 2.75 (m, 1H), 2.58 (s, 6H), 2.47 (m,2H), 2.19 (s, 3H), 2.06 (m, 2H), 1.60 (q, 1H).

(4aS,5aR,12aS)-7-Dimethylamino-3,10,12,12a-tetrahydroxy-9-isoxazol-4-yl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid (Compound CJ)

To 9-iodo-4-dedimethylaminominocycline (2.0 g) was added a DMF (15 mL)previously purged with argon to remove any oxygen, a previously preparedsolution of Na₂CO₃ (784 mg) in water (5.0 mL),dichloro(1,1′bis-diphenylphosphine)(Ferrocene)Pd(0) complexed with DCM(541 mg) and 4-isoxazoleboronic acid pinacol ester (1.08 g). Thereaction was subject to microwave irradiation for duration of 1 minuteat temperature of 100 C. Following, the reaction was added to an aqueoussolution containing acetonitrile (20%) and TFA (0.2%). The solution wasthen filtered through celite to remove the catalyst, loaded onto a C18reverse phase column and the crude product was purified by reverse phaseHPLC (C18, linear gradient 20-40% MeCN in water with 0.1% TFA). Thefractions containing the final product were loaded onto DVB plug, washedwith aqueous HCl (1.0 L, 0.01 N) and eluted with methanol to give theHCl salt of 4-dedimethylamino-9-(isoxazol-4-yl)-minocycline (1000 mg,1.93 mmol, 51%). ¹H-NMR (Bruker DPX300 300 MHz spectrometer, chemicalshifts in ppm with TMS as internal reference at 0 ppm) δ 1.6-1.8 (m,1H), 2.1-2.25 (m, 1H), 2.35-2.7 (m, 3H), 2.9-3.1 (m, 1H), 3.18-3.3 (m,2H), 3.35-3.45 (m, 6H), 8.3 (s, 1H), 9.15 (s, 1H), 9.35 (s, 1H). MWcalcd for C₂₄H₂₃N₃O₈ 481.47, ESIMS found m/z 482 (MH+). Compounds CI,CK, EP, EQ, ER, ES, ET, EU, EV, EW and EX were prepared in this manner.

(4aS,5aR,12aS)-7-Dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-9-pyridin-3-yl-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound CL)

To a 500 mL round bottom flask was added9-iodo-4-dedimethylaminominocycline (2.0 g), NMP (10 mL) and toluene (10mL). The toluene and residual water of stock solution containing9-iodo-4-dedimethylaminominocycline was then removed by rotaryevaporation (5.0 mm Hg, 45° C.) and backfilled with argon to give 10 mLof a 0.37 M stock solution of 9-iodo-4-dedimethylaminominocycline inNMP. To a microwave vial was addedtris(dibenzyldieneacetone)-dipalladium(O) (339 mg), tri-2-furylphosphine(858 mg), CuI (70 mg), stock solution9-iodo-4-dedimethylaminominocycline followed by3-(tri-n-butylstannyl)-pyridine (3 mL). The microwave vial was capped,heated to and maintained at a temperature of 100° C. for 10 minute usinga microwave reactor. The reaction mixture was then cooled to roomtemperature, added to water (1.0 L) and the pH was lowered to 2 usingtrifluoroacetic acid. The solution was then filtered through celite toremove the catalyst, loaded onto a reverse phase column and the crudeproduct was purified by reverse phase HPLC (C18, linear gradient 20-40%MeCN in water with 0.1% TFA). The fractions containing the final productwere loaded onto a DVB plug, washed with aqueous HCl (1.0 L, 0.01 N) andeluted with acetonitrile to give the HC salt (350 mg, 0.66 mmol, 18%).¹H-NMR (Bruker DPX300 300 MHz spectrometer, chemical shifts in ppm withTMS as internal reference at 0 ppm) δ 1.6-1.8 (m, 1H), 2.1-2.25 (m, 1H),2.4-2.7 (m, 3H), 2.95-3.15 (m, 1H), 3.18-3.3 (m, 2H), 3.35-3.45 (m, 9H),8.20-8.29 (m, 1H), 8.3 (s, 1H), 8.91-8.93 (d, 1H, J=5 Hz), 9.01-9.09 (m,1H), 9.3 (s, 1H), MW calcd for C₂₆H₂₅N₃O₇ is 491.50, ESIMS: found m/z492.00 (MH+). Compounds CI, CK, EP, EQ, ER, ES, ET, EU, EV, EW and EXwere prepared in this manner.

(4aS,5aR,12aS)-7-Dimethylamino-3,10,12,12a-tetrahydroxy-9-(2-methyl-2H-pyrazol-3-yl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound EN)

To 9-iodo-4-dedimethyl minocycline (1.5 g, 2.78 mmol, see synthesis ofcompound CC) was added N-methylpyrrolidone (10 mL, previously purgedwith argon to remove any oxygen), a previously prepared solution ofNaCO₃ (584 mg, 5.56 mmol) in water (4.0 mL, also previously purged withargon),1,1′-Bis(diphenylphosphino)-ferrocene)dichloro-palladium(II)complex withdichloromethane 1:1 (400 mg, 0.556 mmol) and1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole(867 mg, 4.17 mmol). The reaction was subject to microwave irradiationfor duration of 2 minutes at temperature of 100° C. Following, thereaction was added to an aqueous solution containing acetonitrile (10%)and TFA (0.2%). The solution was then filtered through celite to removethe catalyst, loaded onto a reverse phase column and purified by HPLC(C18, linear gradient 15-25% acetonitrile in water with 0.1% TFA). Thefractions containing the final product were loaded onto DVB plug, washedwith aqueous HCl (1.0 L, 0.01 N) and eluted with methanol to give theHCl salt of 4-dedimethyl-9-(1-methyl-pyrazole) minocycline (510 mg, 0.96mmol, 35%). ¹H-NMR (Bruker DPX300 300 MHz spectrometer, chemical shiftsin ppm with TMS as internal reference at 0 ppm) δ 1.6-1.8 (m, 1H),2.1-2.25 (m, 1H), 2.35-2.65 (m, 3H), 2.8-3.2 (m, 6H), 3.2-3.3 (m, 1H),3.79 (s, 3H), 6.4 (s, 1H), 7.55 (s, 1H), 7.75 (brs, 1H). MS (electronspray) calcd for C₂₅H₂₆N₄O₇ 494.50, found (M+1) 495.20. Compounds CI,CK, EP, EQ, ER, ES, ET, EU, EV, EW and EX were prepared in this manner.

(4aS,5aR,12aS)-7-Dimethylamino-3,10,12,12a-tetrahydroxy-9-(3-methyl-3H-imidazol-4-yl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound EO)

To a 100 mL round bottom flask was added 9-iodo 4-dedimethyl minocycline(1.5 g, 2.78 mmol, see synthesis of compound CC), N-methylpyrrolidone(10 mL) and toluene (10 mL. The toluene and residual water of the stocksolution containing the 9-iodo 4-dedimethyl minocycline was then removedby rotary evaporation (5.0 mm Hg, 45° C.) and backfilled with argon togive 10 mL of a 0.28 M stock solution of 9-iodo 4-dedimethyl minocyclinein NMP. To a 20 mL microwave vial was addedtris(dibenzyldieneacetone)dipalladium(0) (250 mg, 0.28 mmol),tri-2-furylphosphine (645 mg, 2.8 mmol), CuI (53 mg, 0.28 mmol), and thestock solution of 9-iodo-4-dedimethyl minocycline followed by1-methyl-5-tributylstannanyl-1H-imidazole (2.0 g, 5.56 mmol). Themicrowave vial was capped, heated to 100° C. for 15 minutes using amicrowave reactor. The reaction mixture was then cooled to roomtemperature, added to water (1.0 L) and the pH was lowered to pH 2 usingtrifluoroacetic acid. The solution was then filtered through celite toremove the catalyst, loaded onto a reverse phase column and the crudeproduct was purified by HPLC (C18, linear gradient 10-25% acetonitrilein water with 0.1% TFA). The fractions containing the final product wereloaded onto a DVB plug, washed with aqueous HCl (1.0 L, 0.01 N) andeluted with acetonitrile to give the HCl salt (510 mg, 0.96 mmol, 35%).¹H-NMR (300 MHz spectrometer, chemical shifts in ppm with TMS asinternal reference at 0 ppm) δ 1.6-1.8 (m, 1H), 2.1-2.25 (m, 1H),2.3-2.65 (m, 3H), 2.8-3.0 (m, 7H), 3.4-3.5 (m, 2H), 3.89 (s, 3H), 7.7(d, J=7.4 Hz, 2H), 9.1 (s, 1H). MS (electron spray) calcd for C₂₆H₂₅N₃O₇494.50, found (M+1) 495.20. Compounds CI, CK, EP, EQ, ER, ES, ET, EU,EV, EW and EX were prepared in this manner.

(4aS,5aR,12aS)-9-Cyclopropyl-7-dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound ER)

To a 200 mL round bottom flask was added tetrahydrafuran (THF, 20 mL), astir bar and indium trichloride (InCl₃, 2.21 g, 10.0 mmol). The flaskwas then cooled to −78° C. by placing it in a dry ice bath. A solutionof cyclopropylmagnesiumbromide in THF (60 mL, 0.5 N, 30 mmol) was slowlyadded to the stirred reaction over 5 minutes to generate atricyclopropyl-indium intermediate stock solution. Next, the reactionwas allowed to warm to room temperature. To 9-iodo-4-dedimethylminocycline (2.0 g, 1.2 mmol, see the synthesis of compound CC) wasadded NMP (18 mL) and toluene (18 mL). The toluene and residual water ofstock the stock solution containing 9-iodo-4-dedimethyl minocycline wasthen removed by rotary evaporation (5.0 mm Hg, 45° C.) and backfilledwith argon to give 10 mL of a 0.2 M stock solution of 9-Iodo4-dedimethyl minocycline in NMP. Next, trans-(PdCl₂(PPh₃)₂) (0.378, 0.74mmol) was added to the reaction as well as the abovetricyclopropyll-indium intermediate stock solution (29 mL). The reactionwas heated to 60° C. for 3 hours. The reaction was then added to anaqueous solution (2.0 L) containing acetonitrile (20%) and TFA was addeduntil a pH of 2 was reached. The solution was then filtered throughcelite to remove the catalyst, loaded onto a reverse phase column andpurified by HPLC (C18, linear gradient 20-35% acetonitrile in water with0.1% TFA). The fractions containing the final product were loaded ontoDVB plug, washed with aqueous HCl (1.0 L, 0.01 N) and eluted withacetonitrile to give the HCl salt of 9-cyclopropyl minocycline (450 mg,0.92 mmol, 76%). ¹H-NMR (300 MHz, chemical shifts in ppm with TMS asinternal reference at 0 ppm) S 0.70-0.90)m, 1H), 0.91-1.15 (m, 1H),1.58-1.80 (m, 1H), 2.00-2.40 (m, 2H), 2.40-2.65 (m, 3H), 2.80-3.10 (s,1H), 3.10-3.40 (brm, 8H), 7.45 (s, 1H). MS (electron spray) calcd forC₂₄H₂₆N₂O₇ 454.48, found (M+1) 455.20. Compounds CI, CK, EP, EQ, ER, ES,ET, EU, EV, EW and EX were prepared in this manner.

(4aS,5aR,12aS)-7-Dimethylamino-9-furan-2-yl-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound EU)

MS (ESI+) m/z Theor. Calc. 480.47, Obs. 481.2 (MH+). ¹H NMR (300 MHz,CD₃OD) δ 8.29 (s, 1H), 7.65 (t, 1H), 7.20 (d, 1H), 6.61 (m, 1H), 3.24(d, 1H), 3.14 (dd, 1H), 2.98 (m, 1H), 2.49 (m, 3H), 2.13 (dm, 1H), 1.65(m, 1H).

(4aS,5aR,12aS)-7-Dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-9-phenyl-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound EV)

MS (ESI+) m/z Theor. Calc. 490.509, Obs. 491.20 (MH⁺). ¹H NMR (300 MHz,CD₃OD) δ 7.91 (s, 1H), 7.61 (m, 2H), 7.42 (m, 3H), 3.19 (m, 2H), 2.98(m, 1H), 2.45 (m, 3H), 2.14 (dm, 1H), 1.65 (m, 1H).

(4aS,5aR,12aS)-9-(4-Carbamoyl-phenyl)-7-dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound EW)

MS (ESI+) m/z Theor. Calc. 533.53, Obs. 534.20 (MH⁺). ¹H NMR (300 MHz,CD₃OD) δ 7.96 (m, 3H), 7.75 (m, 2H), 3.16 (m, 2H), 3.01 (m, 1H), 2.52(m, 3H), 2.16 (dm, 1H), 1.67 (m, 1H).

(4aS,5aR,12aS)-9-(5,6-Dihydro-4H-pyran-2-yl)-7-dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound EX)

MS (ESI+) m/z Theor. Calc. 496.51, Obs. 497.20 (MH⁺). ¹H NMR (300 MHz,CD₃OD) δ 7.56 (s, 1H), 5.70 (t, 1H), 4.12 (m, 2H), 3.01 (m, 1H), 2.72(m, 1H), 2.56 (s, 6H), 2.36 (m, 2H), 1.99-2.3 (m, 5H), 1.94 (m, 2H),1.5-1.85 (m, 2H).

(4S,4aS,5aR,12aS)-4,7-Bis-dimethylamino-3,12,12a-trihydroxy-10-oxazol-2-yl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound CM)

To a solution of anhydrous freebase minocycline (11.4 g) in anhydrousTHF under argon (163 mL) at 0° C. was added a 1 M solution of potassiumtert-butoxide (87.5 mL) dropwise. After 45 min,N-phenylbis(trifluoromethanesulfonimide) (18.8 g) was added at once.After 1 hour, the solution was allowed to slowly warm to roomtemperature. After another 2 hours, the solution was slowly poured intoa vigorously stirring solution of 0.1M HCl and Celite. After 15 minutes,the solution was filtered through a large plug of Celite rinsing with0.1M HCl. The water layer was loaded onto a DVB resin for purification.After the solution was loaded, a 0.1 M HCl solution was eluted, thenCH₃CN with 1 mL conc. HCl. The yellow eluent was collected until itbecame colorless. The solution was concentrated under reduced pressureand further dried high vacuum to afford 13.2 g of minocycline10-triflate as a brown solid in 90% yield.

To minocycline 10-triflate (1.0 g) was added DMF (10 mL)dichloro(1,1′bis)(diphenylphosphine)-(ferrocene)Pd (II) complexed withDCM (1.0 g), DIEA (0.50 mL,) and 2-tributylstanyl oxazole. The reactionwas heated to 110° C. for 5 minutes using a microwave reactor. Thereaction mixture was then cooled to room temperature, added to water(0.5 L) and the pH was lowered to 2 using trifluoroacetic acid. Thesolution was then filtered through celite to remove the catalyst, loadedonto a reverse phase column and the crude product was purified byreverse phase HPLC (C18, linear gradient 20-40% MeCN in water with 0.1%TFA). The fractions containing the final product were loaded onto a DVBplug, washed with aqueous HCl (1.0 L, 0.01 N) and eluted withacetonitrile to give the HCl salt of the product (100 mg, 0.71 mmol).ESIMS: m/z 509 (MH+); ¹H-NMR (Bruker DPX300 300 MHz spectrometer,chemical shifts in ppm with TMS as internal reference at 0 ppm) δ1.6-1.8 (m, 1H), 2.3-2.5 (m, 1H), 2.55-2.7 (m, 1H), 2.95-3.15 (m, 7H),3.25-3.3 (m, 6H), 3.35-3.45 (m, 1H), 7.5 (s, 1H), 7.85 (d, J=7.4 Hz,1H), 7.95 (d, J=7.4 Hz, 1H), 8.15 (s, 1H).

(5aR,6aS,7S,10aS)-9-Carbamoyl-4,7-bis-dimethylamino-8.10a,11-trihydroxy-10,12-dioxo-5,5a,6,6a,7,10,10a,12-octahydro-naphthacene-1-carboxylicacid methyl ester (Compound CN)

To a 500 mL flask was added (2.00 g) minocycline-10-triflate and NMP (37mL). To remove residual water from the above reactants, toluene wasadded (37 mL), the flask was placed on the rotary evaporator (35 mm Hg,45° C.) until all the toluene was evaporated. The flask was backfilledwith argon and the contents were then transferred via cannula to a dry250 mL flask. To the 250 mL flask was added dichloromethane adduct ofdichloro(1,1′bis-diphenylphosphine)(Ferrocene)Pd(II) (1.0 g), DIEA (0.50mL) and DIEA (2.60 mL). The flask was placed under vacuum (20 mm Hg) andpurged three times with carbon monoxide. The flask was then heated to60° C. under 1.0 atm of carbon monoxide and let stir for 1 hour. After 1hour, methanol was added (50 mL) and the reaction was allowed to stirfor an additional 1 hour. The reaction mixture was then cooled to roomtemperature, added to water (0.5 L) and the pH was lowered to 2 usingtrifluoroacetic acid. The solution was then filtered through celite toremove the catalyst, loaded onto a reverse phase column and the crudeproduct was purified by reverse phase HPLC (C18, linear gradient 15-35%MeCN in water with 0.1% TFA). The fractions containing the final productwere loaded onto a DVB plug, washed with aqueous HCl (1.0 L, 0.01 N) andeluted with acetonitrile to give the HCl salt of the product (400 mg,0.70 mmol). ESIMS m/z 500 (MH+). ¹H-NMR (Bruker DPX300 300 MHzspectrometer, chemical shifts in ppm with TMS as internal reference at 0ppm) δ 1.6-1.8 (m, 1H), 2.25-2.35 (m, 1H), 2.35-2.7 (m, 1H), 2.95-3.25(m, 14H), 3.25-3.3 (m, 7H), 3.85 (3, 3H), 4.15 (s, 1H), 7.55 (d, J=7.4Hz, 1H), 7.75 (d, J=7.4 Hz, 1H). Compound CO was prepared in thismanner.

(4S,4aS,5aR,12aS)-4,7-Bis-dimethylamino-3,12,12a-trihydroxy-1,11-dioxo-10-(2H-pyrazol-3-yl)-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound EY)

To minocycline-10-triflate (2.0 g, 2.45 mmol, see synthesis of compoundCM) was added NMP (15 mL, previously purged with argon to remove anyoxygen), a previously prepared solution of Na₂CO₃ (1.5 mg, 14 mmol) inwater (5.0 mL, also previously purged with argon) and4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.0 mg, 9.0mmol). The reaction was subject to microwave irradiation for 4 minute attemperature of 125° C. The reaction mixture was then added to an aqueoussolution (2.0 L) containing acetonitrile (10%) and TFA was added until apH of 2 was reached. The solution was then filtered through celite toremove the catalyst, loaded onto a reverse phase column and purified byHPLC (C18, linear gradient 15-25% acetonitrile in water with 0.1% TFA).The fractions containing the final product were loaded onto DVB plug,washed with aqueous HCl (1.0 L, 0.01 N) and eluted with methanol to givethe HCl salt of 10-pyrazole minocycline (150 mg, 0.24 mmol, 10%). ¹H-NMR(300 MHz, chemical shifts in ppm with TMS as internal reference at 0ppm) S 1.6-1.8 (m, 1H), 2.29-2.41 (m, 1H), 2.42-2.60 (m, 1H), 3.01 (s,3H), 3.1 (brs, 4H), 3.18 (brs, 7H), 3.35-3.45 (m, 2H), 4.18 (s, 1H), 6.7(s, 1H), 7.59 (d, J=7.4 Hz, 1H), 7.85 (d, J=7.4 Hz, 1H), 8.15 (s, 1H).MS (electron spray) calcd for C₂₆H₂₉N₅O₆ 507.54. found (M+2) 254.8 (thefound mass represents the doubly charged species, hence the molecularweight found is half the actual molecular weight).

(4S,4aS,5aR,12aS)-10-Cyano-4,7-bis-dimethylamino-3,12,12a-trihydroxy-1,11-dioxo-1,4,4a,5,5a,6,1,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound EZ)

To minocycline-10-triflate (4.0 g, 2.45 mmol, see the synthesis ofcompound CM) was added NMP (30 mL, previously purged with argon toremove any oxygen), Zn(CN)₂ (2.3 g, 19.6 mmol) andtetrakis-(triphenylphosphine)palladium(0) (1.9 g, 1.6 mmol). Thereaction was subject to microwave irradiation for 10 minute at atemperature of 110° C. The reaction mixture was then added to an aqueoussolution (2.0 L) containing acetonitrile (10%) and TFA was added until apH of 2 was reached. The solution was then filtered through celite toremove the catalyst, loaded onto a reverse phase column and purified byHPLC (C18, linear gradient 20-40% acetonitrile in water with 0.1% TFA).The fractions containing the final product were loaded onto DVB plug,washed with aqueous NaOAc (1.0 L, 0.01 N) and eluted with methanol togive the free base of 10-cyano minocycline (250 mg, 0.24 mmol, 10%).¹H-NMR (300 MHz, chemical shifts in ppm with TMS as internal referenceat 0 ppm) δ 1.6-1.8 (m, 1H), 2.05-2.21 (m, 1H), 2.30-2.50 (m, 1H), 2.65(s, 6H), 2.8 (brs, 7H), 3.00-3.20 (m, 1H), 3.25-3.35 (m, 1H), 7.20-7.35(m, 1H), 7.05-7.20 (m, 1H). MS (electron spray) calcd for C₂₄H₂₆N₄O₆466.49, found (M+1) 467.20.

(4S,4aS,5aR,12aS)-4,7-Bis-dimethylamino-3,12,12a-trihydroxy-10-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound FA)

To a 200 mL round bottom flask was added tetrahydrafuran (THF, 40 mL), astir bar and indium trichloride (InCl₃, 4.4 g, 20.0 mmol). The flask wasthen cooled to −78° C. by placing it in a dry ice bath. A solution ofmethylmagnesiumchloride in THF (20 mL, 3.0 N, 60 mmol) was slowly addedto the stirred reaction over 5 minutes to generate a trimethyl-indiumintermediate stock solution. The reaction was then allowed to warm toroom temperature. To minocycline-10-triflate (1.0 g, 0.61 mmol, see thesynthesis of compound CM) was added NMP (10 mL), trans-PdCl₂(PPh₃)₂ (1.0g, 1.4 mmol) and the above trimethyl-indium intermediate stock solution(15 mL). The reaction was subject to microwave irradiation for 4 minutesat a temperature of 110° C. Next, the reaction was added to an aqueoussolution (2.0 L) containing acetonitrile (10%) and TFA was added until apH of 2 was reached. The solution was then filtered through celite toremove the catalyst, loaded onto a reverse phase column and purified byHPLC (C18, linear gradient 15-30% acetonitrile in water with 0.1% TFA).The fractions containing the final product were loaded onto DVB plug,washed with aqueous NaOAc (1.0 L, 0.01 N) and eluted with methanol togive the free base of 10-methyl minocycline (200 mg, 0.44 mmol, 67%).¹H-NMR (300 MHz spectrometer, chemical shifts in ppm with TMS asinternal reference at 0 ppm) δ 1.58-1.8 (m, 1H), 2.30-2.50 (m, 1H),2.45-2.60 (m, 1H), 2.75 (s, 3H), 2.90-3.20 (brm, 8H), 4.19 (s, 1H), 7.48(d, J=7.5 Hz, 1H), 7.89 (d, J=7.5 Hz, 1H). MS (electron spray) calcd forC₂₄H₂₉N₃O₆ 455.51, found (M+1) 456.25.

(4aR,5S,5aR,6R,12aS)-3,5,10,12,12a-Pentahydroxy-6-methyl-9-oxazol-2-yl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound CP)

To a 500 mL round bottom flask was added9-iodo-4-dedimethylaminodoxycycline (3.7 mmol), NMP (10 mL) and toluene(10 mL). The toluene and residual water of stock solution containing9-iodo-4-dedimethylaminodoxycycline was then removed by rotaryevaporation (5.0 mm Hg, 45° C.) and backfilled with argon to give 10 mLof a 0.37 M stock solution of 9-iodo-4-dedimethylaminodoxycycline inNMP. To a microwave vial was addedtetrakis-(triphenylphosphine)palladium(0) (0.37 mmol), the stocksolution 9-iodo-4-dedimethylaminodoxycycline followed by oxazol-2-ylzincin THF at 0.36M (10 mmol). The microwave vial was capped, heated to andmaintained at a temperature of 100° C. for 10 minute using a microwavereactor. The reaction mixture was then cooled to room temperature, addedto water (1.0 L) and the pH was lowered to 2 using trifluoroacetic acid.The solution was then filtered through celite to remove the catalyst,loaded onto a reverse phase column and the crude product was purified byreverse phase HPLC (C18, linear gradient 10-25% MeCN in water with 0.1%TFA). The fractions containing the final product were loaded onto a DVBplug, washed with aqueous HCl (1.0 L, 0.01 N) and eluted withacetonitrile to give the HCl salt. ¹H-NMR (Bruker DPX300 300 MHzspectrometer, chemical shifts in ppm with TMS as internal reference at 0ppm) S 1.56-1.58 (d, 3H, J=6.9 Hz), 2.30-2.55 (m, 2H), 2.7-2.85 (m, 1H),2.9-3.15 (m, 2H), 3.36 (s, 6H), 3.62-3.72 (m, 1H), 7.12-7.14 (d, 1H,J=8.3 Hz), 7.37 (s, 1H), 8.03 (s, 1H), 8.10-8.13 (d, 1H, J=8.3 Hz), MWcalcd for C₂₃H₂₀N₂O₉ 468.42, ESIMS found m/z 469.10 (MH+).

(4S,4aS,5aR,12aS)-4-Dimethylamino-3,10,12,12a-tetrahydroxy-7-(3-methylaminomethyl-furan-2-yl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound CO)

7-iodo-sancycline.TFA (2 g) was combined with palladium (II) acetate (83mg) in methanol (150 mL) in a 2-neck round bottom flask equipped with anargon line. The contents were purged with argon while heating to 70° C.(oil bath temp) for 10 minutes. Separately, sodium carbonate (1.17 g in40 mL water) was purged with argon for 5 minutes prior to addition intothe reaction solution. This was followed by the addition of an argondegassed solution of 3-formyl-furan-2-yl boronic acid (1.0 g in 40 mLDMF). The reaction mixture was stirred at 70° C. in an argon atmospherefor 1-2 hours. Upon completion of reaction (monitored by LC-MS),reaction solution was gravity filtered through celite to removecatalyst, and solvent removed in vacuo. The solid material was dissolvedin methanol, and precipitated using 300 mL of diethyl ether. The solidobtained after filtration was dried overnight in vacuum oven and used assuch for the next step.

A mixture of 7-(3′-formyl-furan-2′-yl)-sancycline (1.02 g), methylaminehydrochloride (0.216 g) and triethylamine (404 μL) in DMF (30 mL) wasstirred for 0.5 hours. Sodium triacetoxyborohydride (1.26 g) was addedand the reaction mixture was stirred for 5 hours. Completion of thereaction was monitored by HPLC/LC-MS. The solvent was then evaporated,and the crude material obtained was redissolved in 5 mL of methanol andprecipitated using 400 mL of diethyl ether. The solid obtained afterfiltration was purified by prep-HPLC (C18, linear gradient 5-35%acetonitrile in water with 0.1% TFA to give a yellow solid, which wasconverted to its HCl salt using saturated MeOH/HCl. ESIMS: m/z 524(MH+). ¹H NMR (300 MHz, CD₃OD): δ 7.67-7.62 (m, 1H), 7.50-7.41 (m, 1H),6.98-6.90 (m, 1H), 6.72-6.69 (m, 1H), 4.08 (s, 1H), 3.96-3.88 (m, 2H),3.20-2.88 (m, 8H), 2.71-2.59 (m, 4H), 2.52-2.39 (m, 1H), 2.15-1.94 (m,1H), 1.60-1.44 (m, 1H). Compounds CS, CT and FB were synthesized in asimilar manner.

2-((6aS,10S,10aS,11aR)-8-Carbamoyl-10-dimethylamino-4,6,6a,9-tetrahydroxy-5,7-dioxo-5,6a,7,10,10a,11,11a,12-octahydro-naphthacen-1-yl)-furan-3-ylmethyl]-methyl-carbamicacid methyl ester (Compound CR)

An amount of 7-(3-methylaminomethyl-furan-2-yl)-sancycline (523 mg) wasdissolved in 10 mL of NMP and methylchloroformate (187 μL) was added tothe solution. The reaction mixture was stirred at room temperature for20 minutes. Upon completion, the product was then precipitated using 250mL of diethyl ether. The solid obtained after filtration was purifiedusing preparative HPLC (C18, linear gradient 10-45% acetonitrile inwater with 0.1% TFA). Fractions were evaporated and the solid obtainedwas then converted to its HCL salt using saturated MeOH/HCl. ESIMS:(m/z) 582 (MH+). ¹H NMR (300 MHz, CD₃OD): δ 7.55-7.50 (m, 1H), 7.48-7.40(d, 1H), 6.94-6.88 (d, 1H), 6.50-6.46 (m, 1H), 4.40-4.11 (m, 2H), 4.06(s, 1H), 3.65-3.52 (m, 3H), 3.20-2.90 (m, 8H), 2.74-2.60 (m, 4H),2.52-2.38 (m, 1H), 2.12-1.88 (m, 1H), 1.62-1.48 (m, 1H). Compounds CS,CT and FB were synthesized in a similar manner.

(4S,12aS)-7-(Bis-trideutromethyl-amino)-4-dimethylamino-9-[(2,2-dimethyl-propylamino)-methyl]-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5.5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound CU)

Dibenzyl azodicarboxylate (132 mmol) was added to trifluoroacetic acid(400 mL) at 5-10° C. and stirred to give a solution. Sancycline (88mmol) was added portionwise while maintaining the temperature below 10°C. The reaction mixture was stirred at 5-10° C. for 7 hours and themixture was concentrated. The residue was dissolved in methanol andhydrogenated at 40 psi H₂ in the presence of 5% Pd/C (20 g) for 3 hours.After completion of reaction, the catalyst was filtered and the methanolwas removed.

The crude 7-aminosancycline was dissolved in methanol. To this solution,deuterated-formaldehyde and 5% Pd/C were added. The reaction mixture wassubjected to hydrogenation with deuterated gas. After the completion ofthe reaction, the crude material was extracted by dichloromethane at pH2and then extracted at pH7. The organic extracts at pH7 were combined andthe solvent was removed. The residue was dissolved in 1N HCl andpurified by prep.HPLC (˜5 g).

Triflic acid (160 mL) was charged into a three-necked round bottom flaskunder nitrogen. The 7-(bis-trideuteromethyl-amino)-sancyclinemono-hydrochloride (0.065 mol) was added portion-wise to the acid,keeping the temperature between 20-25° C. N-hydroxymethylphthalimide wasadded (25.5 g), keeping the temperature between 20-25° C. The reactionmixture was stirred for 1-2 hours and a second portion ofN-hydroxymethylphthalimide was added (6.5 g), still maintaining thetemperature between 20-25° C. Upon completion of the reaction, the acidsolution is added to an ice/water mixture slowly, such that the watertemperature does not rise above 25° C. After stirring for 10 minutes,the product was filtered and washed with water. The solid was dissolvedin acetone (˜0.3 L) and stirred for 5 minutes. Subsequently, thematerial was neutralized to pH 6.0-6.5 with triethylamine. Afterobserving a stable pH for ˜10 minutes, 6 L of water was added to theacetone solution and the yellow solid was fully precipitated out. Thesolid was collected by filtration and washed with water and isopropanol.Upon removal of the solvent, the residue was dried further under reducedpressure at ≤30° C. for 2 days. The product was isolated as a mixture ofthe bis- and tris-alkylated product in an approximately 60:40 ratiobased on HPLC analysis.

A 1 L, three-necked round bottom flask was charged with the intermediate(40 g) under argon. Ethyl alcohol (2 L, anhydrous, 200 proof) was addedwith stirring, and the resulting suspension was cooled to 0-8° C. Asolution of methylamine in ethanol (160 mL, 33%, ˜8 M) was addedgradually such that the reaction temperature remained below 15° C. Thereaction was stirred under an argon atmosphere at 18-28° C. for 15 to 23hours. Upon completion, Celite (16 g) was added; the reaction mixturewas then cooled to 0-5° C. and stirred for 1-2 hours. The resultingsuspension of the phthalamide byproduct and Celite was filtered andwashed with absolute ethanol. The filtrate was charged into a 12 L,three-necked round bottom flask under argon with ice-bath and t-butylmethyl ether (600 L) is added rapidly with stirring, resulting in ayellow suspension. The suspension was stirred for 2 to 3 hours, thenfiltered and washed with THF. The solvent was then removed and theproduct was dried under reduced pressure (under a latex film) and driedovernight under high vacuum at room temperature to obtain 21.8 g of theproduct as a yellow solid.

Methanol (120 mL) was charged in a 1 L pressure bottle under argon,followed by the addition of triethylamine (15 mL). The product from theprevious reaction (20 g) was suspended in this solvent mixture andstirred for 10 minutes.

Trimethylacetaldehyde (20 mL) was added to the suspension over 10minutes. After stirring the resulting solution for 10-15 minutes, 5%Pd/C (10 g) was added. The reaction mixture was purged twice with argon,then three times with H₂. The reaction was stirred under 40-50 psi ofhydrogen pressure for 2-4 hours. Upon completion of the reaction, thesolution was filtered through a celite bed under an argon flow, andwashed with methanol (3×10 mL), prep. HPLC. ESIMS: m/z 563 (MH+).

2,2-Dimethyl-propionic acid(6aS,10S,10aS,11aR)-8-carbamoyl-10-dimethylamino-4,6,6a,9-tetrahydroxy-5,7-dioxo-5,6a,7,10,10a,11.1a,12-octahydro-naphthacen-1-ylmethylcarbamoyloxymethylester (Compound CV)

A mixture of 7-aminomethylsancycline TFA salt (1.5 g) and ammoniumsulfite (100 mg) in a mixture of 40 mL of acetonitrile and 40 mL ofsaturated sodium hydrogen carbonate was purged with argon for 15minutes. A solution of the chloroformate in dry acetonitrile was slowlyadded to the reaction mixture. The reaction mixture was then stirred foradditional 1 hour. The desired material was extracted with severalportions of ethyl acetate. The combined ethyl acetate solution waswashed once with brine and solvent evaporated. The product was obtainedvia C18 column (linear gradient 5-30% acetonitrile in water with 0.1%TFA). ESIMS: m/z 602 (MH+). ¹H NMR (300 MHz, CD₃OD) δ 7.46 (d, 1H), 6.82(d, 1H), 5.72-5.67 (m, 2H), 4.15-4.40 (m, 2H), 4.07 (s, 1H,), 3.2-2.90(m, 9H), 2.40-2.15 (m, 2H), 1.68-1.50 (m, 1H), 1.10 (s, 9H). Compound CWwas also prepared as described above.

(4R,4aS,5aR,12aS)-4-Cyclopentylamino-7-dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound CX)

To a solution of minocycline-2HCI (29.3 g) in DMF (300 mL) was added asolution of hydroxylamine 50% in H₂O (7.98 mL). The solution was heatedto 80° C. for 2 hours while exposed to the air. After cooling to roomtemperature, the solution was diluted with water. The water solution wasfiltered through a plug of DVB resin eluting with a 500 mL gradient ofacetonitrile and water (5%-10%-20%-50%). At the 50% gradient, theproduct eluted as a yellow solution. The solution was concentrated underreduced pressure and further dried under vacuum to afford 12 g as ayellow/orange solid in 45% yield.

To a solution of 4-oximinominocycline (11.1 g) in MeOH (250 mL) and AcOH(7.22 mL), was added 5% Pd on carbon and flushed with H₂. The solutionwas placed under vacuum for three successive cycles. After the finalcycle, the flask was placed under 50 psi of H₂ for 16 hours. Afterflushing the flask with nitrogen, the solution was filtered through aplug of celite, while rinsing with MeOH. The solution was concentratedunder reduced pressure to afford a thick oil. The oil was poured intoisopropanol (1 L) with vigorous stirring and the resulting suspensionwas collected on a sintered funnel while rinsing with cold isopropanol.The 4-amino-minocycline product was further dried under high vacuumovernight to afford 7.6 g as a light brown solid in 71% yield. 1.2 g(1.8 mmol) of 4-amino-minocycline TFA salt was reductively coupled withexcess cyclopentanone (˜10 eq) in the presence of 3 eq. of sodiumtriacetoxyborohydride in 10 ml of DMF. The reaction mixture was stirredat RT for several hrs. The reaction was monitored by analytical HPLC.The final product was isolated via preparative HPLC. ESIMS m/z 498(MH+); ¹H NMR (300 MHz, CD₃OD) δ 7.91 (d, 1H), 7.09 (d, 1H), 4.78 (d,1H), 3.92-3.87 (m, 1H), 3.43 (m, 1H), 3.27 (s, 6H), 3.04-2.94 (m, 2H),2.61-2.51 (m, 1H), 2.25-2.22 (m, 3H), 1.91-1.82 (m, 6H), 1.78-1.74 (m,1H).

(4R,4aS,5aR,12aS)-4-Dimethylamino-7-[2-(2,5-dimethyl-2,5-dihydro-pyrrol-1-yl)-acetyl]-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound CZ)

To a solution of 7-acetylsancycline (2 mmol) in acetic acid and waterwas added hydrogen bromide followed by bromine. The reaction mixture wasstirred for 10 minutes and poured into ether. The solid intermediate wascollected by filtration. The crude product, α-bromoketone, was dissolvedin N-methyl-pyrrolidin-2-one and K₂CO₃, Na₂SO₃ and2,5-dimethylpyrrolidine (1 mL) were added and stirred for 45 minutes.The crude material was purified by prep-HPLC using C18 column (lineargradient 5-30% acetonitrile in water with 0.1% TFA) to give the productas a yellow solid: ESIMS m/z 552 (MH+); ¹H NMR (300 MHz, CD₃OD) δ 8.05(m, 1H), 6.99 (m, 1H), 5.96 (2H, d, J=0.8 Hz), 5.20 (m, 1H), 4.54 (m,2H), 3.78 (m, 1H), 3.23-2.88 (8H), 2.53 (m, 1H), 2.16 (m, 1H), 1.67-1.41(7H). Compound DB was prepared in a similar manner.

Acetic Acid(4S,4aR,5S,5aR,6R,12aS)-9-acetyl-2-carbamoyl-4-dimethylamino-3,10,12,12a-tetrahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacen-5-ylester (Compound CY)

To 10 ml of HF condensed in a polypropylene tube in dry ice, was addedin one portion 1 g of 9-acetyl-doxycycline followed by 10 mL of glacialacetic acid. The reaction mixture was left standing at room temperaturefor overnight. The excess HF was gently removed by slow stream of argon.The residue was then taken by methanol and evaporated to dryness. Thecrude material was directly purified by C18 column (linear gradient1-100% acetonitrile in water with 0.1% TFA). The final product wasisolated as a yellow solid. ESIMS: m/z 529 (MH+); ¹H NMR (300 MHz,CD₃OD) δ 8.08 (1H, d,), 7.1 (1H, d,), 5.25 (m, 1H), 3.79 (s, 1H), 3.01(s, 6H,), 2.90 (m, 2H), 2.65 (s, 3H), 2.22 (s, 3H), 1.36 (d, 3H).

2,2-Dimethyl-propionic acid{allyl-[2-((6aS,10S,10aS,11aR)-8-carbamoyl-10-dimethylamino-4,6,6a,9-tetrahydroxy-5,7-dioxo-5,6a,7,10,10a,11,11a,12-octahydro-naphthacen-1-yl)-2-oxo-ethyl]-carbamoyloxy}-methylester (Compound DA)

7-Acetylsancycline (2 mmol) was dissolved in acetic acid and water.Hydrogen bromide followed by bromine were added and stirred for 10minutes. The intermediate was precipitated from ether and the crudeintermediate, α-bromoketone, was dissolved in N-methyl-pyrrolidin-2-one,and K₂CO₃, Na₂SO₃ and allylamine (0.8 mL) were added and the reactionmixture was stirred for 10 minutes. The solution was precipitated fromether and further purified to give 7-(2′-allylamino-acetyl)-sancyclineas a yellow solid: MS (Mz+1=512).

To a suspension of 7-(2′-allylamino-acetyl)-sancycline (1.2 mmol) andNa₂SO₃ in a mixture of saturated solution of sodium bicarbonate andacetonitrile was added slowly a solution of2,2-dimethylpropionylmethylchloroformate in acetonitrile. The reactionmixture was stirred for 10 minutes. The product was extracted withethyacetate and further purified by prep-HPLC using C18 column (lineargradient 35-45% acetonitrile in water with 0.1% TFA) to give the finalproduct as a yellow solid: ESIMS m/z 670 (MH+); ¹H NMR (300 MHz, CD₃OD)δ 7.92 (m, 1H), 6.94 (m, 1H), 5.84-5.68 (3H), 5.20 (m, 2H), 4.69 (m,1H), 4.43 (m, 1H), 4.11-3.97 (3H), 3.49 (m, 1H), 3.10-2.84 (8H), 2.46(m, 1H), 2.21 (m, 1H), 1.65 (m, 1H), 1.17 (m, 9H). Compound DB wasprepared in a similar manner.

(4S,4aS,5aR,12aS)-7-Cyclopropylaminomethyl-4-dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound DC)

An amount of 1.5 g (2.69 mmol) of 7-formyl-sancycline TFA salt wasreductively aminated with 562 mL of cyclopropylamine in the presence ofsodium triacetoxyborohydride in 10 mL of DMF. The reaction mixture wasstirred at room temperature for several hours. The reaction was followedby C18 column (linear gradient 1-100% acetonitrile in water with 0.1%TFA). ESIMS: (m/z) 484 (MH+). ¹H NMR (300 MHz, CD₃OD) δ 7.64-760 (m,1H), 6.95-6.91 (m, 1H), 4.36 (s, 2H), 4.14 (s, 1H), 3.23 (m, 1H,),3.07-3.00 (m, 8H), 2.87 (m, 1H), 2.50 (m, 1H), 2.4 (m, 1H), 1.55 (m,1H), 0.95 (m, 4H). Compounds DD, DE, DG, DH, DI, DJ and FC were preparedin a similar manner.

(4S,4aS,5aR,12aS)-7-(tert-Butylamino-methyl)-4-dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-1.4.4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound DF)

A mixture of 7-formylsancycline (1 mmol), tert-butylamine (3 mmol), andindium trichloride (0.1 mmol) in DMF was stirred for 1 hour. Sodiumtriacetoxyborohydride (3 mmol) was added and stirred for additional 7hours. The product was purified by C18 column (linear gradient 5-30%acetonitrile in water with 0.1% TFA) to give a yellow solid: ESIMS m/z500 (MH+); ¹H NMR (300 MHz, CD₃OD) δ 7.64 (1H, d, J=8.5 Hz), 6.95 (1H,d, J=8.5 Hz), 4.22 (s, 2H), 4.15 (s, 1H), 3.23-3.01 (9H), 2.49 (m, 1H),2.32 (m, 1H), 1.61 (m, 1H), 1.51 (s, 9H). Compounds DD, DE, DG, DH, DI,DJ and FC were prepared in a similar manner.

(4R,4aS,5aR,12aS)-9-(3,6-Dihydro-2H-pyridin-1-ylmethyl)-4,7-bis-dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound FE)

A solution of 9-formylminocycline (9 mmol) and1,2,3,6-tetrahydropyridine (27 mmol) in 1,2-dichloroethane (120 mL) wasstirred for 40 min. Sodium triacetoxyborohydride (18 mmol) was addedportionwise over 1 hour. The resulting mixture was stirred foradditional 2 hour. The solvent was subsequently reduced and the productwas purified by HPLC using C18 column (linear gradient 10-40acetonitrile in 20 mM aqueous triethanolamine, pH 7.4) to give a yellowsolid: MS (Mz+1=553); ¹H NMR (300 MHz, CD₃OD) δ 8.42 (s, 1H), 6.00 (m,1H), 5.75 (m, 1H), 4.86 (d, 1H, J=3.9 Hz), 4.53 (s, 2H), 3.84 (s, 2H),3.66 (m, 1H), 3.47-3.34 (8H), 3.22-2.98 (8H), 2.61 (m, 2H), 2.45 (m,1H), 2.25 (m, 1H), 1.39 (m, 1H). Compounds FF and FJ were prepared in asimilar manner and compound FL may be prepared in this manner.

(4S,4aS,5aR,12aS)-9-[(3,4-Dihydroxy-benzylamino)-methyl]-4,7-bis-dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2carboxylicacid amide (Compound FD)

M-H=607; ¹H NMR (300 MHz, CD₃OD) δ 7.51 (s, 1H), 6.82-6.9 (m, 3H),4.4-4.4 (m, 4H), 3.06 (s, 6H), 2.75 (s, 6H) 2.63 (sm 2H), 2.43-2.49 (m,4H) 1.8 (m, 2H).

(4S,4aR,5S,5aR,12aS)-4-Dimethylamino-9-(4-fluoro-piperidin-1-ylmethyl)-3,5,10,12,12a-pentahydroxy-6-methylene-1,11-dioxo-1.4.4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound FG)

A mixture 9-formylmethacycline (0.56 g, 1 mmol), and 4-fluoropiperidineHCl (0.28 g, 2 mmol) in DMF (7 mL) was stirred under argon at roomtemperature. To this, triethylamine (202 μL, 2 mmol) was added and thereaction mixture was stirred at room temperature for 1 hour. Sodiumcyanoborohydride (0.19 g, 3 mmol) was then added and the reactionmixture was stirred at room temperature for another 2 hours. The DMF wasremoved and the crude material was purified using preparative HPLC (C18,linear gradient 10-40% acetonitrile in water with 0.1% TFA). The yellowsolid obtained after evaporation was converted to its HCl salt usingsaturated solution of methanol-HCl MS (ESI+) m/z 557.57, obs. 558.30(MH⁺); ¹H NMR (300 MHz, CD₃OD) δ 7.70 (d, J=7.2 Hz, 1H), 7.24 (d, J=7.2Hz, 1H), 5.65 (d, J=3.5 Hz, 1H), 5.50 (d, J=3.5 Hz, 1H) 4.55 (s, 1H),4.40 (s, 2H), 3.88 (m, 1H), 3.67 (m, 1H), 3.55-3.32 (m, 3H), 3.02-2.78(m, 7H), 2.40-1.91 (m, 4H). Compounds FF and FJ were prepared in asimilar manner and compound FL may be prepared in this manner.

(4S,4aR,5S,5aR,12aS)-4-Dimethylamino-3,5,10,12,12a-pentahydroxy-9-[(methoxy-methyl-amino)-methyl]-6-methylene-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound FH)

A mixture 9-formylmethacycline (0.56 g, 1 mmol), and N, O-dimethylhydroxylamine HCl (0.19 g, 2 mmol) in DMF (10 mL) was stirredunder argon at room temperature for 1 hour. Sodium cyanoborohydride(0.09 g, 1.5 mmol) was then added and the reaction mixture was stirredat room temperature for another 1 minute.

The DMF was removed and the crude material was purified usingpreparative HPLC (C18, linear gradient 15-40% acetonitrile in water with0.1% TFA). The yellow solid obtained after evaporation was converted toits HCl salt using saturated solution of methanol-HCl MS (ESI+) m/z515.51, obs. 516.25 (MH+); ¹H NMR (300 MHz, CD₃OD) δ 7.69 (d, J=7.2 Hz,1H), 7.18 (d, J=7.2 Hz, 1H), 5.65 (d, J=3.5 Hz, 1H), 5.48 (d, J=3.5 Hz,1H) 4.60 (s, 1H), 4.10 (s, 2H), 3.91 (m, 1H), 3.70 (m, 1H), 3.58 (s,3H), 3.12-2.94 (m, 6H), 2.81 (s, 3H), 2.10 (s, 1H). Compounds FF and FJwere prepared in a similar manner and compound FL may be prepared inthis manner.

(4S,4aS,5aR,2aS)-4,7-Bis-dimethylamino-9-[N″-(2,2-dimethyl-propionyl)-hydrazinomethyl]-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound FI)

To solution of 9-formylminocycline (1.21 g, 2.50 mmol) in anhydrousdichloroethane (25 mL) at room temperature was added pivaloylhydrazide(0.465 g, 4.00 mmol). After 16 hours, the solution was concentratedunder reduced pressure. The crude product was redissolved in acetic acidat room temperature and borane trimethylamine complex (0.191 g, 2.63mmol) was added. After 12 hours, the solution was poured into 2% TFAwater. The water solution was loaded onto a DVB column for solid phaseextraction and the product was isolated by eluting with 1% TFA/CH₃CN.The crude product was further purified by preparatory HPLC (C18, lineargradient 10-60% acetonitrile in water with 0.1% TFA) to afford 0.47 g in32% yield as a yellow solid. ¹H NMR (300 MHz, CD₃OD) δ 8.15 (s, 1H),4.49-4.44 (m, 2H), 4.12 (s, 1H), 3.48-3.35 (m, 1H), 3.23-2.90 (m, 16H),2.57-2.46 (m, 1H), 2.33-2.25 (m, 1H), 1.70-1.53 (m, 1H), 1.12 (s, 9H).LC/MS (MH⁺) 586. Compounds FF and FJ were synthesized in a similarmanner and compound FL may be synthesized in a similar manner.

(4S,4aS,5aR,12aS)-4,7-Bis-dimethylamino-3,10,12,12a-tetrahydroxy-9-(4″-tert-butyl-semicarbazido-1″-methyl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound FK)

To solution of 9-formylminocycline (1.21 g, 2.50 mmol) in anhydrousdichloroethane (25 mL) at room temperature was addedt-butylsemicarbazide (0.391 g, 3.00 mmol). After 16 hours, the solutionwas concentrated under reduced pressure. The crude product wasredissolved in acetic acid (8.3 mL) at room temperature and boranetrimethylamine complex (0.191 g, 2.63 mmol) was added. After 12 hours,the solution was poured into 2% TFA water. The water solution was loadedonto a DVB column for solid phase extraction and the product wasisolated by eluting with 1% TFA/CH₃CN. The crude product was furtherpurified by preparatory HPLC (C18, linear gradient 10-60% acetonitrilein water with 0.1% TFA) to afford 0.53 g in 35% yield as a yellow solid.¹H NMR (300 MHz, CD₃OD) δ 8.06 (s, 1H), 4.42-4.39 (m, 2H), 4.11 (s, 1H),3.32-3.14 (m, 4H), 3.07-2.91 (m, 7H), 2.56-2.43 (m, 1H), 2.37-2.28 (m,1H), 1.72-1.57 (m, 1H), 1.20 (s, 9H). LC/MS (MH⁺) 601. Compounds FF andFJ were synthesized in a similar manner and compound FL may besynthesized in a similar manner.

(4aS,5aR,12aS)-7-Dimethylamino-3,10,12,12a-tetrahydroxy-9-(3-isopropyl-[1,2,4]oxadiazol-5-yl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound FM)

To a 500 mL flask was added (4.00 g, 8.60 mmol) 4-dedimethylamine-9-iodominocycline free base (see the synthesis of compound CC), NMP (50 mL),N-hydroxysuccinimide (3.9 g, 38 mmol). To the reaction flask was added astir bar, tetrakis(triphenylphosphine)palladium(0) (2.00 g, 1.67 mmol)and DIEA (3.0 mL, 1.7 mmol). The flask was placed under vacuum (20 mmHg) and purged 3 times with carbon monoxide. The flask was then heatedto 60° C. under 1.0 atm of carbon monoxide and was stirred for 1 houruntil the 4-dedimethlyamine-9-iodo minocycline was consumed and a peakfor the corresponding 9-NHS-ester 4-dedimethylamino minocyclineintermediate [(M+1) of 556 M/Z] was formed as observed by LCMS. TheNHS-ester intermediate was then reacted withN′-hydroxy-2-methylpropanimidamide (2.0 g, 19.6 mmol) at roomtemperature for 2 hours to give the noncyclized intermediate [(M+1) of543 M/Z] as determined by LCMS. The noncyclized intermediate wasisolated by adding it to 50 mL acetonitrile followed by dilution of thereaction mixture with water to a total volume of 2.0 L. Subsequently,the water was adjusted to a pH of 2 using trifluoroacetic acid. Theaqueous solution was then filtered and loaded onto a plug ofdivinylbenzene resin and purified (10-60% MeCN, 0.1% TFA) to give 1 g ofcrude noncyclized intermediate. To noncyclized-intermediate (2.0 g, 3.7mmol) in a 500 mL round bottom flask was added NMP (80 mL) and toluene(80 mL). To prevent hydrolysis during the subsequent cyclization step,residual water was removed from the noncyclized intermediate bysubjecting it to rotary evaporator (5 mm Hg, 45° C.) until all thetoluene/water was evaporated. Next, the flask was backfilled with argonand diisopropylamine (2 mL, 1.13 mmol) was added. To facilitatecyclization the contents were heated to 125° C. for 8 minutes viamicrowave irradiation. The reaction was then added to acetonitrile,diluted with water to a final volume of two liters and trifluoroaceticacid was added to a final pH of 2. The solution was then filteredthrough celite to remove the catalyst, loaded onto a reverse phasecolumn and the crude product was purified by HPLC (C18, linear gradient30-40% acetonitrile in water with 0.1% TFA). The fractions containingthe final product were loaded onto a DVB plug, washed with aqueous HCl(1.0 L, 0.01 N) and eluted with methanol to give the HCl salt of9-(3-isopropyl-1,2,4-Oxadiazoyl)-4-dedimethylamino minocycline (280 mg,0.53 mmol, 12%). ¹H-NMR (300 MHz, chemical shifts in ppm with TMS asinternal reference at 0 ppm) δ 1.47 (d, J=7.5 Hz, 6H), 1.55-1.75 (m,1H), 2.0-2.2 (m, 1H), 2.15-2.6 (m, 3H), 2.6-2.9 (m, 7H), 3.05-3.19 (m,1H), 3.20-3.45 (m, 3H), 8.00 (s, 1H). ESI-MS (electron spray) calcd. forC₂₆H₂₈N₄O₈ 524.54, found (M+1), 525.25. Compound FN was also synthesizedin a similar manner.

(4aS,5aR,12aS)-7-Dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-9-propionyl-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound FR)

A reaction flask (in case of propyne, a pressure reactor was used forreaction) was charged with, 9-iodo-4-dedimethylaminominocycline (10 g,18.51 mmol, see the synthesis of compound CC), palladium (II) acetate(0.415 g, 1.85 mmol), CuI (0.705 g, 3.70 mmol), and Pd(PPh₃)₄ (2.14 g,1.85 mmol) and the acetylene (4 equiv). The solvent was added to thereaction mixture, followed by the acetylene reagent and the Et₃N and thereaction flask was purged with argon for 1 minute. The reaction flaskwas heated to 75° C. and allowed to stir until the starting material hasbeen consumed (typical reaction time is between 5 minutes and 1 hour),as indicated by sampling the reaction at regular intervals via LCMS. Thereaction mixture was then filtered warm, through a bed of Celite andwashed with 4×50 mL of MeCN. The combined filtrate was evaporated todryness, which was further dried under high vacuum for 18 hours toafford the crude acetylene-substituted product. It was used withoutpurification for the next step.

To the flask containing the dried product from the previous step wasadded the TFA solution (100 mL) and stirred at room temperature for 5minutes, followed by stirring at 80° C. for 5 min. An 80% solution ofH₂SO₄ (100 mL) was freshly prepared and added (while hot) to thereaction mixture over approximately 60 seconds. The reaction turned intoa solution, after a momentary appearance of some precipitate. Thereaction mixture was stirred at 75° C. until the reaction was confirmedto be complete by LCMS monitoring. The reaction mixture was poured into800 mL of ice-water and allowed to stir for 20 minutes. The suspensionwas filtered over a bed of Celite and the precipitate was washed with6×100 mL portions of dilute HCl (ca. 2-3%) and/or dilute formic acid(ca. 2-3%). The yellow filtrate was filtered again through a 0.22μ fritand purified by prep-HPLC (C18, linear gradient 25-55% acetonitrile inwater with 0.2% formic acid, 280 nm) carefully to remove a closelyeluting impurity (detected only by MS, obs, m/z 557.2). The combinedpure fractions were diluted three times with water and the solution wasloaded onto a clean DVB column, washed with water containingapproximately 1% HCl (ca. 2 L) and eluted into methanol. The methanolsolution was evaporated to dryness and further dried under high vacuumfor 18 hours to afford 3.17 g of the desired product as an HCl salt. MS(ESI+) m/z Theor. Calc. 470.47, obs. 471.20 (MH+). ¹H NMR (300 MHz,CD₃OD) δ 8.25 (s, 1H), 3.13 (m, 3H), 2.98 (m, 1H), 2.50 (m, 3H), 2.12(dm, 1H), 1.68 (m, 1H), 1.17 (t, 3H).

(4aS,5aR,12aS)-7-Dimethylamino-9-(3,3-dimethyl-butyryl)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound FS)

MS (ESI+) m/z Theor. Calc. 512.55, Obs. 513.25 (MH+). ¹H NMR (300 MHz,CD₃OD) δ 8.16 (s, 1H), 3.18 (m, 1H), 3.07 (s, 2H), 2.97 (m, 1H), 2.50(m, 3H), 2.12 (dm, 1H), 1.66 (m, 1H), 1.06 (s, 9H).

(4aS,5aR,12aS)-7-Dimethylamino-9-(3-dimethylamino-propionyl)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound FT)

MS (ESI+) m/z Theor. Calc. 513.54, Obs. 514.30 (MH⁺). ¹H NMR (300 MHz,CD₃OD) δ 8.38 (s, 1H), 3.69 (m, 2H), 3.55 (m, 2H), 3.22 (m, 1H), 3.00(m, 7H), 2.51 (m, 3H), 2.14 (dm, 1H), 1.69 (m, 1H).

(4aS,5aR,12aS)-7-Dimethylamino-9-(3-dimethylamino-propyl)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1.4.4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound FU

The 9-acetylene minocycline intermediate (0.870 g) prepared fromN,N-dimethyl-propargylamine was dissolved in MeOH (20 mL) and stirredunder a hydrogen atmosphere (50 psi) in presence of 10% Pd/C (0.092 g)for 3 hours, when LCMS monitoring of the reaction confirmed completionof the reaction. The reaction mixture was filtered over a bed of Celite,washed with 2×10 mL of MeOH and the combined filtrate was evaporated todryness. The product was purified using preparative HPLC (C18, lineargradient 15-55% acetonitrile in water with 0.2% formic acid, 280 nm).The pure product fractions were concentrated on a DVB column, convertedinto HCl salt, and eluted in pure methanol. The methanol solution wasevaporated to dryness, and further dried under high vacuum for 12 hoursto afford the desired product as its HCl salt (0.180 g). MS (ESI+) m/z499.56, obs. 500.30 (MH⁺). ¹H NMR (300 MHz, CD₃OD) δ 7.93 (s, 1H), 3.18(m, 4H), 2.98 (m, 1H), 2.90 (s, 6H), 2.82 (t, 2H), 2.48 (m, 3H), 2.11(m, 3H), 1.65 (m, 1H).

(4aS,5aR,12aS)-7-Dimethylamino-9-ethyl-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound FV)

In a 2-necked, 100 mL flask, 9-iodo-4-dedimethylamino-minocyclinefreebase (3.0 g, 5.56 mmol, see synthesis of compound CC) and Pd(PPh₃)₄(0.291 g, 0.569 mmol) were charged and the flask was degassed by vacuumfollowed by argon three times. To the flask, 30 mL of THF was added andthe solution/suspension was allowed to stir for 2-3 minutes. A 0.24 Msolution of Et₃In (prepared from the reaction of ethylmagnesium chloride(3 equiv) and InCl₃) (23.1 mL, 5.55 mmol) was added to the reactionmixture. The flask was equipped with a reflux condenser and the reactionmixture was allowed to reflux (oil bath temp=85° C.). After 1 hour, areaction aliquot showed (by LCMS) the consumption of the startingmaterial. The reaction flask was removed from the oil bath and allowedto cool to room temperature. The reaction mixture was diluted with 60 mLof water and treated with TFA to adjust the pH to 2. The organic solventwas reduced by rotary evaporation. The residual mixture was then dilutedwith 25 mL of water containing 1% TFA and filtered through Celite. TheCelite layer was washed with 3×20 mL portions of 1% TFA/water. Theresulting yellow solution was purified using preparative HPLC (C18,linear gradient 20%-50% acetonitrile in water with 0.1% TFA, 280 nm).The main fractions were diluted 4× with water, loaded onto a DVB column,washed first with 1% HCl in water and then eluted in MeOH. The yellowMeOH solution was then evaporated to dryness, dissolved again inMeOH/dil HCl and evaporated to dryness. It was further dried under highvacuum for 16 hours to afford 1.272 g of pure material. MS (ESI+) r/zTheor. Calc. 442.46, obs. 443.20 (MH+). ¹H NMR (300 MHz, CD₃OD) δ 7.78(s, 1H), 3.18 (m, 4H), 2.95 (m, 1H), 2.73 (q, 2H), 2.43 (m, 3H), 2.11(dm, 1H), 1.64 (m, 1H), 1.24 (t, 3H).

(4aS,5aR,12aS)-7-Dimethylamino-3,10,12,12a-tetrahydroxy-9-isopropyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound FW)

MS (ESI+) m/z Theor. Calc. 456.49, obs. 457.20 (MH+). ¹H NMR (300 MHz,CD₃OD) δ 7.73 (1H), 3.11 (m, 1H), 2.94 (m, 1H), 2.43 (m, 3H), 2.11 (dm,1H), 1.63 (m, 1H), 1.26 (m, 6H).

(4aS,5aR,12aS)-7-Dimethylamino-9-(2,2-dimethyl-propyl)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound FX)

MS (ESI+) m/z Theor. Calc. 484.54, obs. 485.25 (MH+). ¹H NMR (300 MHz,CD₃OD) δ 7.69 (s, 1H), 3.11 (dd, 1H), 2.96 (m, 1H), 2.66 (m, 2H), 2.45(m, 3H), 2.12 (dm, 1H), 1.65 (m, 1H), 0.96 (s, 9H).

(4aS,5aR,12aS)-7-Dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-9-trifluoromethyl-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound FY)

A 100 mL 2-necked flask was charged with a stir bar,9-iodo-4-dedimethylamino-minocycline (1.86 g, 3.43 mmol, see thesynthesis of compound CC), CuI (0.721 g, 3.78 mmol), HMPA (10 mL) andmethyl-2,2-difluoro-2-(sulfonyl)-acetate (1.2 mL, 9.43 mmol). Thereaction mixture was purged with argon, and stirred at 80° C. withfrequent monitoring of the reaction mixture with LCMS. After 30 minutes,another 1.2 mL of methyl-2,2-difluoro-2-(sulfonyl)-acetate was added tothe reaction mixture and allowed to stir at that temperature. Upon theconsumption of the starting material as indicated by LCMS, the reactionmixture was cooled to room temperature and poured into 200 mL of water.The suspension was stirred for 2 hours, filtered, washed with 5×20 mLportions of water, and air dried. The crude material was dissolved inMeCN (containing 2% TFA), diluted with water 5 times and purified bypreparative HPLC (C18, linear gradient 20%-55% acetonitrile in waterwith 0.1% TFA, 280 nm). The pure product fractions were concentrated ona DVB column, converted into HCl salt, eluted with MeOH and themethanolic solution was evaporated to dryness. It was further driedunder high vacuum for 16 hours to afford 0.241 g of the desired product.MS (ESI+) m/z Theor. Calc. 482.41, obs. 483.35 (MH*). ¹H NMR (300 MHz,CD₃OD) δ 8.16 (s, 1H), 3.00 (m, 1H), 2.5 (m, 3H), 2.13 (dm, 1H), 1.66(m, 1H).

(4R,4aS,5aR,12aS)-4,7-Bis-dimethylamino-9-ethyl-.10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-aphthacene-2-carboxylicacid amide (Compound FZ)

¹H NMR (300 MHz, CD₃OD) δ7.30 (s, 1H), 3.78 (d, 1H), 3.44 (m, 1H), 3.01(br.s, 6H), 2.99-2.85 (m, 2H), 2.68 (q, 2H), 2.62 (s, 6H), 2.28-2.02 (m,1H), 1.63 (q, 1H), 1.21 (t, 3H). LCMS (m/z): 486 (MH+).

(4S,4aR,5S,5aR,12aS)-4-Dimethylamino-3,5,10,12,12a-pentahydroxy-6-methylene-1,11-dioxo-9-pyridin-2-yl-1,4,4a,5.5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound GA)

An amount of 9-iodo-methacycline (569 mg, 1 mmol), Pd (PPh₃). (115 mg,0.1 mmol), and copper iodide (38 mg) were taken in 20 mL of DMF. To thispyridin-2-yl tributyl stannane (441 mg, 1.2 mmol) was added and thereaction mixture was stirred at room temperature for 3 hours. Completionof the reaction was monitored using LCMS. The solvent was then removedunder vacuum and the crude material was purified using preparative HPLC(C18, linear gradient 10-40% acetonitrile in water with 0.1% TFA). Theyellow solid obtained after evaporation was converted to its HCl saltusing saturated solution of methanol-HCl MS (ESI+) m/z 519.50, obs.520.25 (MH⁺); ¹H NMR (300 MHz, CD₃OD) δ 8.80 (d, J=3.4 Hz, 1H), 8.51 (t,1H), 8.28 (d, J=7.2 Hz, 1H), 7.99 (d, J=7.2 Hz, 1H), 7.89 (t, 1H), 7.35(d, J=7.2 Hz, 1H), 5.71 (s, 1H), 1H), 5.15 (s, 1H) 4.55 (s, 1H), 3.89(m, 1H), 3.70 (m, 1H), 3.06-2.88 (m, 7H).

(4S,4aS,5aR,12aS)-7-Chloro-4-dimethylamino-9-ethyl-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound GB)

A 100 mL 2 or 3 neck round-bottom flask with reflux condenser wascharged with anhydrous InCl₃ (1.21 g, 4.05 mmol) and dried under vacuumwith a heat gun. After the flask cooled to ambient temperature andflushed with argon, anhydrous THF (24.0 mL) was added. The solution wascooled to −78° C. and EtMgBr (12.2 mL, 12.2 mmol) as a 1 M solution inTHF was added. After 15 minutes, the solution was allowed to slowly warmto room temperature to form a clear heterogeneous solution. The reactionflask was added 7-chloro-9-iodosancycline (2.07 g, 3.60 mmol) andPd(t-Bu₃)₂ (0.092 g, 0.180 mmol). The solution was heated to refluxunder argon until complete (approximately 1 hour). After cooling toambient temperature, the solution was quenched with MeOH (0.5 mL) andpoured into a stirring cold solution of 1M HCl (0.3 L). After 1 hour,the solution was filtered through a pad of Celite rinsing with water.The water solution was loaded onto a DVB column for solid phaseextraction and the product was isolated by eluting with 1% TFA/CH₃CN.The crude product was further purified by preparatory HPLC (C18, lineargradient 15-65% acetonitrile in water with 0.1% TFA, to afford 1.31 g in76% yield as a yellow solid. ¹H NMR (300 MHz, CD₃OD) δ 7.38 (s, 1H),4.05 (s, 1H), 3.29-3.20 (m, 1H), 3.06-2.93 (m, 8H), 2.69-2.50 (m, 2H),2.34-2.18 (m, 2H), 1.72-1.57 (m, 1H), 1.16 (t, J=7.1 Hz, 3H). LC/MS(MH⁺) 477.

(4aS,5aR,12aS)-3,10,12,12a-Tetrahydroxy-7-isopropyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound GC)

A 100 mL 2-necked flask was charged with a stir bar,7-iodo-4-dedimethylamino-sancycline (1.5 g, 3.02 mmol), palladium (II)acetate (0.068 g, 0.31 mmol), Pd(PPh₃)₄ (0.349 g, 0.31 mmol), andmethanol (20 mL). The reaction mixture was then purged with argon. Thesuspension was first treated with an aqueous solution of Na₂CO₃ (0.962g, 0.91 mmol), and then with a DMF solution of 2-propenyl-boronic acid(prepared from a modified procedure from Tet. Lett. 2001, 42, 4083-4085)(0.391 g, 4.55 mmol in 3 mL DMF). The reaction mixture was stirred at70° C. for 2 hours and monitored with LCMS. Upon the completion ofreaction, it was precipitated with water (200 mL), the resultingprecipitate was filtered, washed with water and air-dried. The crudeproduct was purified by preparative HPLC (C18, linear gradient 30%-75%acetonitrile in water with 0.1% TFA, 280 nm). The pure product fractionswere evaporated to dryness and the resulting material was used as suchfor the next step. The product from the previous step (0.670 g) wasdissolved in methanol, treated with 0.080 g of 10% Pd/C, and stirredunder a hydrogen atmosphere (50 psi) for 3 hours. Upon the consumptionof the starting material, the reaction mixture was filtered through abed of Celite, washed the filter with 2×20 ml, methanol portions and thecombined organic solution was evaporated to dryness. It was purified bypreparative HPLC (C18, linear gradient 30%-75% acetonitrile in waterwith 0.1% TFA, 280 nm). The pure fractions were combined and evaporatedto dryness. Upon further drying under high vacuum for 18 hours, 0.252 gof desired product was isolated. MS (ESI+) m/z Theor. Calc. 413.42, obs.414.10 (MH⁺). ¹H NMR (300 MHz, CD₃OD) δ 7.42 (d, 1H), 6.77 (d, 1H), 2.79(m, 1H), 2.29 (m, 3H), 2.02 (m, 1H), 1.58 (m, 1H), 1.16 (dd, 6H).

(4aS,5aR,12aS)-3,10,12,12a-Tetrahydroxy-7-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound GD)

MS (ESI+) m/z Theor. Calc. 385.37, obs. 386.15 (MH⁺). ¹H NMR (300 MHz,CD₃OD) δ 7.26 (d, 1H), 6.67 (d, 1H), 2.98 (dd, 1H), 2.79 (m, 1H), 2.44(m, 2H), 2.20 (m, 4H), 2.01 (m, 1H), 1.56 (m, 1H).

(4aS,5aR,12aS)-3,10,12,12a-Tetrahydroxy-7-oxazol-2-yl-1,11-dioxo-1.4.4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound GE)

MS (ESI+) m/z Theor. Calc. 438.39, obs. 439.20 (MH⁺). ¹H NMR (300 MHz,CD₃OD) δ 8.03 (s, 1H), 7.96 (d, 1H), 7.36 (s, 1H), 6.96 (d, 1H), 3.64(m, 1H), 2.84 (m, 1H), 2.48 (m, 3H), 2.02 (m, 1H), 1.59 (m, 1H).

(4aS,5aR,12aS)-7-Acetyl-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound GF)

MS (ESI+) n/z Theor. Calc. 413.38, obs. 414.20 (MH+). ¹H NMR (300 MHz,CD₃OD) δ 7.96 (d, 1H), 6.87 (d, 1H), 3.45 (dd, 1H), 2.78 (m, 1H), 2.52(s, 3H), 2.41 (m, 3H), 1.99 (dm, 1H), 1.55 (m, 1H).

(4S,4aS,5aR,12aS)-7-Cyclopropyl-4-dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound GH)

A 1000 mL 2 or 3 neck round-bottomed flask with reflux condenser wascharged with anhydrous InCl₃ (12.1 g, 40.5 mmol) and dried under vacuumwith a heat gun. After the flask cooled to ambient temperature and wasflushed with argon, anhydrous THF (240 mL) was added. The solution wascooled to −78° C. and c-PrMgBr (244 mL, 122 mmol) as a 0.5 M solution inTHF was added. After 15 minutes, the solution was allowed to slowly warmto room temperature to form a clear heterogeneous solution. To thereaction flask was added 7-iodosancycline (19.4 g, 36.0 mmol) andPd(t-Bu₃)₂ (0.920 g, 1.80 mmol). The solution was heated to reflux underargon until complete (approximately 1 hour). After cooling to ambienttemperature, the solution was quenched with MeOH (1 mL) and poured intoa stirring cold solution of 1M HCl (3 L). After 1 hour, the solution wasfiltered through a pad of Celite, while rinsing with water. The watersolution was loaded onto a DVB column for solid phase extraction and theproduct was isolated by eluting with 1% TFA/CH₃CN. The crude product wasfurther purified by preparatory HPLC (C18, linear gradient 20-60%acetonitrile in water with 0.1% TFA, to afford 11.8 g in 72% yield as ayellow solid. ¹H NMR (CD₃OD) δ 7.18 (d, J=7.4 Hz, 1H), 6.61 (d, J=7.4Hz, 1H), 3.99 (s, 1H), 3.34 (dd, J=9.1, 2.8 Hz, 1H), 3.05-2.84 (m, 10H),2.32-2.09 (m, 2H), 0.86-0.75 (m, 2H), 0.51-0.37 (m, 2H). LC/MS (MH+)455.

(4aS,5aR,12aS)-7-Dimethylamino-3,12,12a-trihydroxy-10-methoxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound GI)

A solution of minocycline (6 g) in THF (80 mL) was cooled to −68° C. andtreated with solid tert-BuO⁻K⁺ (5.37 g) and allowed to stir for 60minutes. The suspension was allowed to warm to room temperature over aperiod of 1.5 hours. The reaction was cooled to 4° C., treated withMeOTs (5.5 mL) and allowed to stir to warm up slowly to roomtemperature. The reaction was approximately 75% complete after 5 hours,when it was poured into 0.75 L of ether, stirred for 1 hour at roomtemperature, filtered (slow filtration) and washed with ether (3×200mL). Upon drying overnight under vacuum, 12.2 g of crude (dark green)solid was isolated. All the solid was dissolved first in 10% MeCN/water(ca. 500 mL) and the solution (dark green) was acidified with dilute HCl(pH=2). The solution was then diluted with triethanolamine-aqueousbuffer (pH=7.4) and the pH was adjusted to 7.3-7.4 with aqueous NaHCO₃solution and 3-4 mL of triethanolamine. The solution was filtered overCelite and the filtrate was diluted with water (2 L). The material waspurified using preparative HPLC (C18, linear gradient 15-35%acetonitrile in 20 mM aqueous triethanolamine and TFA, pH 7.4). Theproduct fractions were immediately acidified with TFA. The purefractions were combined, the pH adjusted with aqueous NaHCO₃ solution to7.3, and then diluted with triethanolamine/TFA buffer (pH=7.4). Thetriethanolamine was washed away with water after loading the productfractions (diluted 3 times with water) onto a DVB column. The productwas eluted with pure methanol and the yellow solution evaporated todryness. The product was further dried under high vacuum for 18 hours toafford 1.37 g (isolated) of yellow solid. It was used for the next stepwithout further purification.

To a solution of 10-methoxyminocycline (1.3 g) in THF (15 mL, warmedbriefly), was added Mel (1.5 mL, excess) and allowed to stir at roomtemperature. When the LC/MS monitoring of the reaction mixture indicatedconsumption of the starting material, the reaction mixture was treatedwith 160 mL of t-BME. The resulting yellow suspension was stirred for 2hours, and the precipitate was filtered, washed with 3×100 mL of t-BMEportions and dried under vacuum (air-aspirator). The solid was furtherdried for 3 h under high vacuum. Isolated 1.6 g of yellow powder.

The de-dimethyl-amination step was carried out using standard reductionconditions, described earlier for the synthesis of 4-dedimethylaminominocycline. The product was purified using preparative HPLC (C18,linear gradient 15-35% acetonitrile in water containing 0.2% formicacid) affording 0.245 g of product as its HCl salt. MS (ESI+) m/z Theor.Calc. 428.43, obs. 429.15 (MH+). ¹H NMR (300 MHz, CD₃OD) δ 7.91 (d, 1H),7.27 (d, 1H), 3.94 (s, 3H), 3.04 (m, 1H), 2.83 (m, 1H), 2.44 (m, 3H),2.15 (dm, 1H), 1.63 (m, 1H).

(4aS,5aR,12aS)-9-Benzoyl-7-dimethylamino-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound GK)

To suspension of 9-iodo-4-dedimethylamino-minocycline (DDAM, freebase, 6g, 11.1 mmol), Pd(PPh₃)₄ (1.37 g, 1.185 mmol), N-hydroxysuccinimide(6.51 g, 56.56 mmol) in NMP (50 mL), was added di-isopropylethylamine (9mL). The reaction mixture was degassed (vacuum/argoncycle) and then thereaction flask was heated to 80° C. in an oil bath. Abutyl-rubber-balloon was filled with CO and connected to the reactionflask. The reaction mixture was then degassed and refilled with CO threetimes and allowed to stir under a CO atmosphere. The reaction wasmonitored with LC-MS and when >98% of the 9-I-4-DDAM was consumed (withthe formation of 9-hydroxysuccinimide-ester of4-dedimethylamino-minocycline, obs, m/z 556), the reaction mixture wastransferred (using a cannula) into a dry 200 mL flask containing sodium4-methylbenzenethiolate (3.78 g, 25.6 mmol) and a stir bar. Thisreaction mixture was stirred first at 80° C. for 1.5 hours and then atroom temperature for 2 hours. The LC-MS indicated the formation of thedesired 9-Tol-S-ester (obs m/z=565). The reaction mixture was thenfiltered through a bed of Celite and the filtrate was poured into water(500 mL). The resulting suspension was stirred for 30 minutes and thenacidified with dilute HCl (pH=1). The product was extracted into EtOAc(3×350 mL) and the organic layer was washed with 3×400 mL portions ofwater, followed by brine (200 mL). The organic layer was dried overanhydrous Na₂SO₄ and evaporated to dryness to afford 8.3 g of crudethioester. The crude product was used in the next step.

To a suspension of crude9-(4-methylphenyl)thiocarboxylacyl-4-dedimethylamino-minocycline (1.2 g,2.12 mmol), copper(I)-thiophenecarboxylate (0.650 g, 3.4 mmol),Pd₂(dba)₃ (0.106 g, 0.116 mmol), and P(2-furyl)₃ (0.196 g, 0.84 mmol) inanhydrous THF (10 mL) under argon was added a solution of previouslyprepared Ph₃In (16 mL of 0.29 M solution in THF, prepared earlier). Thereaction mixture was heated at 90° C. for 2 hours, while monitoring atregular intervals. If the reaction appeared stalled, fresh catalysts andreagent were added to the reaction mixture and continued to stir at 90°C. until the reaction was complete. The reaction mixture was filteredover a bed of Celite and the Celite bed was washed with 3×10 mL portionsof THF. The combined filtrate and the washings were evaporated todryness (a thick oil). The residue was dissolved in MeCN (100 mL)containing 5 mL of TFA and diluted with water (500 mL). The suspensionwas filtered over a bed of Celite and the filtrate was purified usingpreparative HPLC (C18, linear gradient 25%-45% acetonitrile in watercontaining 0.2% formic acid). The product fractions were combined,diluted with ca. 1.8 L of water and loaded onto a DVB column (0.5″×3″diam.). The product was first washed with 8-10% methanol/water (600 mL),and then with a solution containing ca. 1% HCl/20% MeOH/water (ca. 1200mL, total volume). The product was eluted in MeOH. The yellow solutionwas collected, evaporated to dryness, suspended/dissolved in MeOH (20mL) 15% HCl (2 mL) and evaporated to dryness again. The product wasfurther dried overnight under high vacuum to afford 0.412 g of theproduct. MS (ESI+) m/z Theor. Calc. 518.519, obs. 519.25 (MH⁺). ¹H NMR(300 MHz, CD₃OD) δ 8.02 (s, 1H), 7.82 (m, 2H), 7.65 (m, 1H), 7.51 (m,2H), 3.16 (dd, 1H), 3.06 (m, 1H), 2.51 (m, 3H), 2.15 (dm, 1H), 1.68 (m,1H).

(4S,4aS,5aR,12aS)-4-Dimethylamino-9-[(methoxy-methyl-amino)-methyl-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound GL)

An amount of 7-bromo-9-formyl-sancycline (1.92 mmol) was combined withN,O-dimethyl-hydroxylamine HCl salt (3.84 mmol) and DMA (8 mL) andstirred under an argon atmosphere at room temperature for 1.5 hours.Sodium cyanoborohydride (2.3 mmol) was added and the reaction wasmonitored by LC/MS. Reaction was complete within 10 minutes. Thereaction mixture was triturated in diethyl ether (300 mL), and filteredto give 1.3 g of 7-bromo-9-methoxyaminomethyl sancycline. An amount of7-bromo-9-methoxyaminomethyl sancycline (0.88 mmol) was combined withammonium formate (8.83 mmol), Pd(dppf)₂CH₂Cl₂ (0.0883 mmol), InCl₃(0.442 mmol), and NMP (7 mL) in a microwave vial, and placed in themicrowave on high absorbance for 5 minutes at 100° C. The reactionmixture was poured into water (400 mL with 0.1% TFA) and was filteredthrough celite. The crude product was purified by prep-HPLC using a C-18column (linear gradient 10-40% acetonitrile in water with 0.1% TFA).ESI-MS: (MH+)=488. ¹H NMR (300 MHz, CD₃OD) δ 7.64 (1H, d, J=9 Hz), 6.90(1H, d, J=6 Hz), 4.67 (m, 2H), 4.11 (s, 1H), 3.98 (m, 3H), 3.17 (m, 4H),2.97 (m, 9H), 2.61 (m, 1H), 2.23 (m, 1H), 1.61 (m, 1H). Compound GM wasalso synthesized in a similar manner.

(4S,4aS,5aR,12aS)-7-Bromo-4-dimethylamino-3,10,12,12a-tetrahydroxy-9-iodo-1,11-dioxo-1,4,4a,5.5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound GN)

To a solution of sancycline TFA (53.2 g, 101 mmol) in TFA (400 mL) at 0°C. was added powdered NBS (85.4 g, 303 mmol) in portions over a 15minutes period. After 2 hours, solid NIS (45.4 g, 202 mmol) was addedand stirred for another 5 hours at 0° C. The solution was poured into avigorously stirring solution of 10% Na₂SO₃ (500 mL) at 0° C. where thepi was adjusted pH=7.5 with solid NaOAc. After 30 minutes, the resultingsuspended product was collected on a fine fritted funnel rinsing withwater. The crude product was redissolved in a solution of THF (300 mL)with DVB (approx. 200 g) then poured into a vigorously stirring solutionof 0.5 M HCl. The suspension was poured onto a prepared DVB column andafter loading the product was eluted with MeOH with 1% HCl. The productin solution was concentrated under reduced pressure then dried underhigh vacuum to afford 57.5 g as a yellow solid in 92% yield. ¹H NMR(CD₃OD) δ 8.02 (s, 1H), 3.98 (s, 1H), 3.17-2.78 (m, 9H), 2.29-2.06 (in,2H), 1.62-1.46 (m, 1H). LC/MS (MH⁺) 620. Compound GQ was synthesized ina similar manner.

(4S,4aS,5aR,12aS)-7-Bromo-4-dimethylamino-9-formyl-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound GO)

An oven dried 500 mL 3-neck flask with internal thermometer was chargedwith 9-iodo-7-bromosancycline (18.9 g, 30.5 mmol), powdered anhydrousNaOAc (5.00 g, 61.0 mmol), Pd(PPh₃)₄ (3.52 g, 2.50 mmol) and anhydrousNMP at (102 mL). The flask was purged with carbon monoxide by bubblingthe solution for 20 minutes, then a large balloon of carbon monoxide wasaffixed to the top of the flask to maintain a positive pressure. Thereaction flask was heated to 70° C. At 30 minutes past obtained reactiontemperature, Bu₃SnH (9.02 mL, 33.6 mmol) was added via syringe pump at arate of 2.26 mL per hour for a total of 4 hours. After completion ofreaction, the reaction was cooled to ambient temperature and then pouredinto a stirring solution of 2.5% TFA/H₂O (1000 mL) with Celite andpotassium fluoride (17.7 g, 305 mmol). After 15 minutes, the solutionwas filtered through a plug of Celite rinsing with 1% TFA/H₂O. Thecombined water solution was loaded onto a previously prepared column ofDVB resin (7×15 cm packed DVB column). After loading, a solution of 1%TFA/H₂O (approx. 1 L) was eluted then a gradient of CH₃CN/water with 1%TFA was eluted to obtain the desired product. The fractions containingproduct were concentrated under reduced pressure to afford 14 g in 71%yield as a TFA salt. ¹H NMR (CD₃OD) (exists as methyl hemi-acetal) δ7.74 (s, 1H), 5.56 (s, 1H), 4.03 (s, 1H), 3.22-3.15 (m, 3H), 3.02-2.85(m, 3H), 2.38-2.27 (m, 1H), 2.19-2.08 (n, 1H), 1.66-1.50 (m, 1H). LC/MS(MH+) 523, 521 due to Br. Compound GQ was synthesized in a similarmanner.

(4S,4aS,5aR,12aS)-4-Dimethylamino-9-(4-fluoro-piperidin-1-ylmethyl)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound GP)

A mixture of 7-bromo-9-formyl sancycline (1.07 g, 2 mmol), InCl₃ (0.02g, 0.2 mmol), and 4-fluoropiperidine HCl (0.84 g, 6 mmol) in DMF (20 mL)was stirred under argon at room temperature. To this triethylamine (808μL, 6 mmol) was added and the reaction mixture was stirred at roomtemperature for 1 hour. Sodium cyanoborohydride (0.19 g, 3 mmol) wasthen added and the reaction mixture was stirred at room temperature foranother 2 hours. The completion of the reaction was monitored by LCMS.The reaction was quenched by adding water/0.1% TFA (1 L), the pH of thesolution was adjusted to 2 by adding TFA. The resulting solution wasthen passed through celite and washed with 200 mL of water. The waterlayer was then loaded onto a DVB column, washed with water and thedesired product was eluted with methanol. Solvent was evaporated to givea brown-yellow solid which was used without further purification for thenext step.

An amount of 7-bromo-9-(4′-fluoropiperdinyl)-aminomethyl sancycline(1.07 g) was taken in 50 mL of methyl alcohol. To this Pd/C (5%, 250 mg)was added and the reaction mixture was hydrogenated at 45 psi for 6hours. The reaction was filtered through celite and the yellow filtratewas evaporated to dryness to give a yellow solid, which was purifiedusing preparative HPLC (C18, linear gradient 7-25% acetonitrile in waterwith 0.1% TFA). The organic solvent was reduced and the water layer wasloaded onto a DVB column to remove water. The desired product was elutedwith methanol. The yellow solid obtained was converted to its HCl saltusing saturated solution of methanol-HCl. MS (ESI+) m/z 529.56, obs.530.00 (MH⁺); ¹H NMR (300 MHz, CD₃OD) δ 7.67 (d, J=8.6 Hz, 1H), 6.92 (d,J=8.5 Hz, 1H), 5.05 (m, 1H), 4.41 (s, 2H), 4.10 (s, 1H), 3.65-3.44 (m,2H), 3.30-2.91 (m, 10H), 2.62 (t, 1H), 2.41-1.92 (m, 5H), 1.52 (m, 1H).Compound GQ was synthesized in a similar manner.

[4-((6aS,10R,10aS,11aR)-8-Carbamoyl-10-dimethylamino-4,6,6a,9-tetrahydroxy-5,7-dioxo-5,6a,7,10,10a,11,11a,12-octahydro-naphthacen-1-yl)-phenyl]-carbamicacid 2-fluoro-ethyl ester (Compound GR)

A solution of 7-iodosanscycline trifluoroacetate (654.43 mg, 1 mmol),Pd(PPh₃)₄ (115.6 mg, 0.1 mmol), and palladium (II) acetate (22.5, 0.1mmol) in 20 mL methanol was purged with argon for 10 minutes. A solutionof sodium carbonate (424 mg, 4 mmol) in 5 mL water was added and themixture was purged for additional 5 minutes. A solution of 4-nitrophenylboronic acid (0.33 g, 2 mmol) in DMF (5 mL) was purged with argon andadded to the mixture. The reaction mixture was heated to 65° C. andstirred at the same temperature for 3 hours. The reaction mixture wascooled and filtered through celite pad. The filtrate was taken, solventevaporated and the crude product was precipitated from ether, which wasused for the next step without any purification.

An amount of 7-(4′-Nitro-phenyl)-sancycline (1.0 g) was taken in 50 mLof methyl alcohol. To this Pd/C (5%, 250 mg) was added and the reactionmixture was hydrogenated at 45 psi for 3 hours. The reaction wasfiltered through celite and the yellow filtrate was evaporated todryness to give a yellow solid, which was used for the next step withoutany purification.

An amount of 7-(4′-amino-phenyl)-sancycline (2.02 g, 2 mmol), was takenin 25 mL of NMP. To this solution 2-fluoroethyl chloroformate (1.01 g, 8mmol) was added and the reaction mixture was stirred at room temperaturefor 1 hour. The reaction mixture was then poured into 2 L of water with0.1% TFA and was then filtered through celite to give a clear yellowsolution, which was then purified using preparative HPLC (C18, lineargradient 10-50% acetonitrile in 20 mM aqueous triethanolamine, pH 7.4).The organic solvent was reduced and the water layer was loaded onto aDVB column, which was washed with 6 L of water. The desired product wasthen eluted with methanol. The yellow solid obtained was converted toits HCl salt using saturated solution of methanol-HCl. MS (ESI+) n/z595.57, obs. 596.35 (MH⁺); ¹H NMR (300 MHz, CD₃OD) δ 7.49 (d, J=7.2 Hz,2H) 7.38 (d, J=8.5 Hz, 1H), 7.15 (d, J=7.2 Hz, 2H), 6.87 (d, J=8.5 Hz,1H), 4.81 (m, 1H), 4.72 (m, 1H), 4.56 (m, 1H), 4.46 (m, 1H), 4.32 (m,1H), 3.17 (s, 3H), 3.00-2.73 (m, 6H), 2.50 (t, 1H), 1.89 (m, 1H), 1.51(m, 1H).

(4S,4aS,5aR,12aS)-4-Dimethylamino-9-(2-dimethylamino-acetylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-7-pyridin-2-yl-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (Compound GU)

An amount of 7-iodo-9-nitrosancycline (0.58 g, 1 mmol), Pd(PPh₃)₄ (0.11g, 0.1 mmol), CuI (0.038 g, 0.2 mmol) were taken in anhydrous DMF (20mL), and purged with argon for 5 minutes. To this solution2-pyridinyl-stannane (0.44 g, 1.2 mmol) was added and the reactionmixture was stirred at room temperature for 2 hours. The reaction wasthen poured into 1 L of water/0.1% TFA solution and was filtered throughcelite and washed with water/0.1% TFA (100 mL). The water layer was thenloaded to a DVB column. The product was isolated at 15-20% acetonitrileand the solvent was evaporated to give a yellow-brown powder, which wasused for the next step without further purification.

An amount of 7-pyridin-2-yl-9-nitrosancycline (200 mg) was taken in 20mL of methyl alcohol. To this Pd/C (5%, 20 mg) was added and thereaction mixture was hydrogenated at 40 psi for 2 hours. The reactionwas filtered through celite and the yellow filtrate was evaporated todryness to give a yellow solid, which was used for the final stepwithout further purification.

An amount of 7-pyridin-2-yl-9-aminosancycline (0.50 g, 1 mmol) was takenin 7 mL of NMP. To this solution N,N-dimethylglycyl chloride (2 mmol)was added and the reaction mixture was stirred at room temperature for10-60 minutes. The reaction mixture was poured into 1 L of water with0.1% TFA and then filtered through celite to give a clear yellowsolution, which was then purified using preparative HPLC (C18, lineargradient 10-25% acetonitrile in 20 mM aqueous triethanolamine, pH 7.4).MS (ESI+) m/z 591.61, obs. 592.35 (MH+); ¹H NMR (300 MHz, CD₃OD) δ 8.92(d, J=4.5 Hz, 1H), 8.70 (t, 1H), 8.51 (s, 1H), 8.13 (m, 2H), 4.30 (s,2H), 4.11 (s, 1H), 3.21-2.90 (m, 14H), 2.68 (m, 2H), 2.13 (m, 1H), 1.55(m, 1H). Compounds GT and GV were also synthesized in a similar manner.

[((5aR,6aS,7S,10aS)-9-Carbamoyl-7-dimethylamino-1,8,10a,11-tetrahydroxy-10,12-dioxo-4-pyridin-2-yl-5,5a,6,6a,7,10,10a,12-octahydro-naphthacen-2-ylcarbanoyl)-methyl]-trimethyl-ammonium(Compound GS)

MS (ESI+) m/z 606.65, obs. 606.35 (M+) ¹H NMR (300 MHz, CD₃OD) δ 8.90(d, J=4.5 Hz, 1H), 8.69 (t, 1H), 8.46 (s, 1H), 8.12 (m, 2H), 4.49 (s,2H), 4.09 (s, 1H), 3.40 (s, 9H), 3.21-2.90 (m, 7H), 2.67 (m, 2H), 2.12(m, 1H), 1.52 (m, 1H).

Example 2. Anti-Bacterial Activity

In this example, the gram (+) and gram (−) antibacterial activities ofthe tetracycline compounds used in the methods of the invention wereassessed.

Gram (−) and gram (+) antibacterial minimum inhibitory concentration(MIC) values (μg/mL) were obtained using CLSI methodology foranti-bacterial susceptibility testing. On each day of testing, serialdilutions of compounds were prepared in microdilution plates using aTecan robotic workstation. Mueller Hinton broth cultures ofrepresentative sensitive and resistant gram negative strains were grownor adjusted to match the turbidity of a 0.5 McFarland standard. 1:200dilutions were made in an appropriate broth (cation supplemented MuellerHinton broth) to allow a final inoculum of 1×10⁵ cfu. Plates wereincubated at 35° C. in ambient air for 18-24 hours, were readspectrophotometrically and checked manually for evidence of bacterialgrowth. The lowest dilution of compound that inhibited growth wasrecorded as the MIC. Lysed horse blood was used to supplement broth fortesting S. pneumoniae. The MIC's for each compound were assessed againstS. aureus, S. pneumoniae, P. acnes, E. coli and B. theta. The resultsare shown in Table 3. Good antibacterial activity (e.g., less than about4 μg/mL) is indicated by “***,” modest antibacterial activity (betweenabout 4 and 8 μg/mL) is indicated by “**,” or weak or no antibacterialactivity (greater than about 8 μg/mL) is indicated by “*.” The symbol“-” indicates that no data was obtained.

TABLE 3 Compound S. aureus S. pneumoniae P. acnes P. acnes E. coli E.coli B. thetaiotaomicron Code RN450 157E - Strep ATCC 6919 ATCC 11827ATCC 25922 MG 1655 ATCC 29741 A *** *** *** *** *** ** * B *** *** ****** * * ** C *** *** *** *** * * * D *** *** ** ** * * * E *** *** ****** ** * * F *** *** *** *** * * ** G *** *** ** ** ** * * H *** *** **** * * * J *** *** *** *** *** *** *** K *** *** *** *** *** *** *** L*** *** *** *** *** *** *** M *** *** *** *** *** ** ** N *** *** ****** *** ** *** O *** *** *** *** *** *** *** P *** *** *** *** ** ** **Q *** *** *** *** ** ** ** R *** *** *** *** * * ** S *** *** **** * * * T *** *** *** *** *** *** ** U *** *** *** *** *** ** ** V ****** *** *** * * ** W *** *** *** *** * * * X *** *** *** *** *** *** ***Y *** ** *** *** * * * Z *** *** *** *** ** ** * AA *** *** *** *** **** ** AB *** *** *** *** ** * ** AC *** *** *** *** ** * ** AD *** ****** *** ** ** * AE *** *** *** *** ** * ** AF *** *** *** *** ** ** ***AL *** *** *** *** ** ** ** AM ** ** *** *** * * ** AN *** *** *** ****** *** ** DR *** *** *** *** ** * ** DT *** *** *** *** ** ** ** DV ****** *** *** ** * ** DW *** *** *** *** *** * ** DX *** *** *** *** ** ***** DY *** ** *** *** * * ** DZ *** *** *** *** ** * *** EA *** ** ****** * * ** EB *** ** *** *** * * ** EC *** *** — — * * — ED *** *** ****** ** ** *** GL ** ** ** ** * * ** GM *** *** *** ** * * ** Doxycycline*** *** *** *** *** *** ** Minocycline *** *** *** *** *** *** **

Example 3: Toxicity Profile

In this example, the cytotoxicity of the tetracycline compounds used inthe methods of the invention were assessed.

Mammalian cell cytotoxicity was assessed to evaluate potential in vivorisks associated with the tetracycline compounds of the invention. Asoluble, non-toxic redox dye (“Resazurin”; Alamar Blue) was used toassess a tetracycline compound's effect on cellular metabolism. At theonset of the experiment, cultures of mammalian COS-1 or CHO cells werewashed, trypsinized, and harvested. Cell suspensions were prepared,seeded into 96-well black-walled microtiter plates, and incubatedovernight at 37° C., in 5% CO₂ and approximately 95% humidity. On thenext day, serial dilutions of test drug were prepared under sterileconditions and transferred to cell plates. Plates were then incubatedunder the above conditions for 24 hours. Following the incubationperiod, the media/drug was aspirated, and 50 μL of resazurin was added.Plates were then incubated under the above conditions for 2 hours andthen in the dark at room temperature for an additional 30 minutes.Fluorescence measurements were taken (excitation 535 nm, emission 590nm) and toxic effects in treated versus control cells were comparedbased on the degree of fluorescence in each well. The results are shownin Table 4. Minocycline and doxycycline toxicity scores are shown forcomparison. Compounds which showed cytotoxicity at concentrations ofless than about 35 μg/mL are indicated by “***,” compounds which showedcytoxicity at concentrations between about 35 and 75 μg/mL are indicatedby “**,” and compounds that showed minimal or no cytoxicity areindicated by “*” (e.g., at concentrations greater than about 75 μg/mL).

TABLE 4 COS-1 CHO Cytotoxicity Cytotoxicity IC₅₀ IC₅₀ Compound (μg/mL)(μg/mL) Minocycline * * Doxycycline * * A *** *** B *** *** C * * D * *E * * F *** *** G * * H * * J *** *** K *** *** L *** *** M * * N * * O** *** P * * Q *** *** R *** *** S * * T *** *** U * ** V *** *** W * *X *** *** Y * * Z * * AA * * AB *** *** AC *** *** AD *** *** AE * ***AF ** ** AL *** *** AM * * AN * * DR ** ** DV *** *** DW * * DX *** **DY * * DZ *** *** EA * * ED *** *** GL ** ** GM *** ***

Example 4: Phototoxic Potential

In this example, the phototoxic potential of the tetracycline compoundsused in the methods of the invention was assessed. In particular, 3T3fibroblast cells were harvested and plated at a concentration of 1×10⁵cells/mL and the plates were incubated overnight at 37° C., in 5% CO₂and approximately 95% humidity. On the following day the medium wasremoved from the plates and replaced with Hanks' Balanced Salt Solution(HBSS). Drug dilutions were made in HBSS and added to the plates. Foreach compound tested, a duplicate plate was prepared that was notexposed to light as a control for compound toxicity. Plates were thenincubated in a dark drawer (for controls), or under UV light (meterreading of 1.6-1.8 mW/cm) for 50 minutes. Cells were then washed withHBSS, fresh medium was added, and plates were incubated overnight asdescribed above. The following day neutral red was added as an indicatorof cell viability. The plates were then incubated for an additional 3hours. Cells were then washed with HBSS and blotted on absorbent paperto remove excess liquid. A solution of 50% EtOH, 10% glacial acetic acidwas added and after 20 minutes incubation, and the plate's absorbance at535 nm was read using a Wallace Victor 5 spectrophotometer. Thephototoxicity reflected the difference between the light-treated andcontrol cultures. The results are given in Table 5. Results for thetetracycline derivative COL-3, as well doxycycline and minocycline areshown for comparison. Compounds which showed phototoxicity are indicatedby “****” (e.g., less than 5 μg/mL), compounds which showed moderatephototoxicity are indicated by “***” (e.g., greater than about 5 μg/mLand less than about 25 μg/mL), compounds which showed some phototoxicityare indicated by “**” (e.g., greater than about 25 μg/mL and less thanabout 75 μg/mL) and compounds that showed minimal or no phototoxicityare indicated by “*” (e.g., greater than about 75 μg/mL).

TABLE 5 Compound Dark Tox50 UV Tox50 Code (μM) (μM) Minocycline * *Doxycycline * *** COL-3 ** **** A * *** B * * C * ** D * ** E * ** F *** G * ** H * * J * * K * * L * **** M * * N * * O * ** P * ** Q * *R * * S * ** T * * U * * V * * W * ** X * *** Y * * Z * * AA * * AB **** AC * * AD * * AE * ** AF * * AL * *** AM * * AN * * DR * * DV * **DW * * DX * ** DY * * DZ * *** EA * * ED * * GM * **

Example 5. Half-Life Determination of the Oxidation

In this example, the half-life of minocycline and a tetracyclinecompound of the invention were assessed under oxidative conditions, asdescribed in Nilges, et al. (Nilges M, Enochs W, Swartz H. J. Org. Chem.1991, 56, 5623-30). Not to be limited by theory, it is believed that thetissue staining may be caused oxidative instability. The tetracyclinecompounds were subjected to accelerated oxidation in a continuous-flowmicroreactor using a 15 molar excess of sodium periodate at pH 11 and22° C. Aliquots of each reaction mixture were quenched at various timepoints with ascorbic acid and the disappearance of each compound wasdetermined by RP-HPLC. Pseudo first-order rate constants and t_(1/2)values were obtained from the plots of log (Ao−At/Ao) versus time, whereAo is the HPLC area determined for each compound at time=0 and At is theHPLC area at time=t. The results indicated that minocycline had ahalf-life for oxidation of 8.2 seconds, while compound B had a half-lifefor oxidation of 495 seconds.

Example 6: In Vivo Anti-Bacterial Activity with S. aureus Model

In this example, the in vivo anti-bacterial activity of the tetracyclinecompounds used in the methods of the invention were assessed.

Groups of five mice were injected intraperitoneally with a lethal doseof S. aureus RN450 in a medium of mucin. Mice were evaluated at 24 hoursto determine survival. Untreated animals experienced 100% mortality.Subcutaneous treatment with a single dose of minocycline, doxycycline orthe test compound resulted in 100% survival. In some instances, a doseresponse study was performed with the compound such that a PD₅₀ (a doseof compound that protects 50% of the animals) could be calculated. Theresults are shown in Table 6.

TABLE 6 Dose Percent PD50 Compound (mg/kg) Survival (mg/kg) Untreated — 0 (0/5) — Minocycline 5 100 (5/5) 0.72 Doxycycline 5 100 (5/5) 0.13 A 5100 (5/5) — C 5 100 (5/5) — P 5 100 (5/5) 0.13 Q 5 100 (5/5) 0.45 V 1.4 W 1.08 AA 5 100 (5/5) — AD 4.54 AF 0.23 DV 1.1  DW 0.48 DX 0.58 0.58 DZ1.11

Example 7: In Vivo Anti-Inflammatory Activity with RatCarrageenan-Induced Paw Edema Inflammatory Model

To asses the anti-inflammatory potential of the tetracycline compoundsused in the methods of the invention, the tetracycline compounds wereassessed in a model of carrageenan induced rat paw inflammation. Themodel used a sub-plantar injection of carrageenan in the rat to inducean inflammatory response. The test compound or saline (control) wasadministered IP 30 minutes before a subplantar injection of carrageenan(1.5 mg/0.1 mL). Paw volume was measured (mm²) before subplantarinjection and again 3 hours after the injection of carrageenan using aplethysmometer. The results are shown in FIGS. 1 and 2. Significantdifferences as determined by a Kruskal-Wallis One Way ANOVA are notedbetween the inflammation of the untreated controls versus treatedanimals (p=0.5)

FIG. 1 compares the modulation of carregeenan induced inflammation ofdoxycycline with various doses of compound A. Doxycycline exhibited a50% effective concentration (EC₅₀) at approximately 50 mg/kg, whilecompound A exhibited improved activity.

FIG. 2 compares the modulation of carregeenan induced inflammation ofminocycline compared with various doses of compound P. Minocyclineexhibited an EC₅₀ at approximately 50 mg/kg, while compound P exhibitedsimilar or improved activity.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, patents, and patentapplications cited throughout this application are hereby incorporatedby reference. The appropriate components, processes, and methods ofthose patents, applications and other documents may be selected for thepresent invention and embodiments thereof.

1-391. (canceled)
 392. A method for treating a tetracycline responsivestate in a subject, comprising administering to said subject aneffective amount of a tetracycline compound of formula (VIIA):

wherein: X is CR⁶*CR^(6*′); R^(5*′) is hydrogen; R^(5*′) is hydrogen orhydroxyl; R^(6*′) is hydrogen or alkyl; R^(6*′) is hydrogen; R^(7′) isalkylamino; and R^(9m*) is oxazolyl or isoxazolyl or a pharmaceuticallyacceptable salt thereof, such that said subject is treated.